Use of multipotent adult stem cells in treatment of myocardial infarction and congestive heart failure

ABSTRACT

The invention provides isolated stem cells of non-embryonic origin that can be maintained in culture in the undifferentiated state or differentiated to form cells of multiple tissue types. Also provided are methods of isolation and culture, as well as therapeutic uses for the isolated cells in cardiac indications.

RELATED APPLICATIONS/PATENTS

This application is a continuation of U.S. application Ser. No.11/151,689, filed Jun. 13, 2005 (allowed), which is acontinuation-in-part of U.S. application Ser. No. 10/963,444, filed Oct.11, 2004, which is a continuation-in-part of U.S. application Ser. No.10/048,757 filed Feb. 1, 2002 which is a U.S. National Stage Applicationof PCT/US00/21387 filed Aug. 4, 2000 and published in English as WO01/11011 on Feb. 15, 2001, which claims priority under 35 U.S.C. 119(e)from U.S. Provisional Application No. 60/147,324 filed Aug. 5, 1999 and60/164,650 filed Nov. 10, 1999. U.S. application Ser. No. 10/963,444,filed Oct. 11, 2004, is also a continuation-in-part of U.S. applicationSer. No. 10/467,963 filed on Aug. 11, 2003 which is a U.S. NationalStage Application of PCT/US02/04652 filed Feb. 14, 2002 and published inEnglish as WO 02/064748 on Aug. 22, 2002, which claims priority under 35U.S.C. 119(e) from U.S. Provisional Application No. 60/268,786 filedFeb. 14, 2001; 60/269,062 filed Feb. 15, 2001; 60/310,625 filed Aug. 7,2001; and 60/343,836 filed Oct. 25, 2001, which applications andpublications are herein incorporated by reference.

FIELD OF THE INVENTION

The field of the invention is alleviating the symptoms of heart damage,especially resulting from myocardial infarction, by administeringmultipotent adult stem cells.

BACKGROUND OF THE INVENTION

Stem Cells

The embryonal stem (ES) cell has unlimited self-renewal and candifferentiate into all tissue types. ES cells are derived from the innercell mass of the blastocyst or primordial germ cells from apost-implantation embryo (embryonal germ cells or EG cells). ES and EGcells have been derived from mouse, and, more recently, from non-humanprimates and humans. When introduced into blastocysts, ES cells cancontribute to all tissues. A drawback to ES cell therapy is that, whentransplanted in post-natal animals, ES and EG cells generate teratomas.

ES (and EG) cells can be identified by positive staining with antibodiesto SSEA1 (mouse) and SSEA4 (human). At the molecular level, ES and EGcells express a number of transcription factors specific for theseundifferentiated cells. These include Oct-4 and rex-1. Also found arethe LIF-R (in mouse) and the transcription factors sox-2 and rox-1.Rox-1 and sox-2 are also expressed in non-ES cells. A hallmark of EScells is the presence of telomerase, which provides these cells with anunlimited self-renewal potential in vitro.

Oct-4 (Oct 3 in humans) is a transcription factor expressed in thepregastrulation embryo, early cleavage stage embryo, cells of the innercell mass of the blastocyst, and in embryonic carcinoma (EC) cells(Nichols J, et al (1998) Cell 95:379-91), and is down-regulated whencells are induced to differentiate. Expression of Oct-4 plays animportant role in determining early steps in embryogenesis anddifferentiation. Oct-4, in combination with Rox-1, causestranscriptional activation of the Zn-finger protein Rex-1, also requiredfor maintaining ES undiffereniated (Rosfjord E, Rizzino A. (1997)Biochem Biophys Res Commun 203:1795-802; Ben-Shushan E, et al (1998) MolCell Biol 18:1866-78). In addition, sox-2, expressed in ES/EC, but alsoin other more differentiated cells, is needed together with Oct-4 toretain the undifferentiated state of ES/EC (Uwanogho D et al (1995) MechDev 49:23-36). Maintenance of murine ES cells and primordial germ cellsrequires LIF.

The Oct 4 gene (Oct 3 in humans) is transcribed into at least two splicevariants in humans, Oct 3A and Oct 3B. The Oct 3B splice variant isfound in many differentiated cells whereas the Oct 3A splice variant(also previously designated Oct 3/4) is reported to be specific for theundifferentiated embryonic stem cell. See Shimozaki et al (2003)Development 130:2505-12.

Adult stem cells have been identified in most tissues. Hematopoieticstem cells are mesoderm-derived and have been purified using cellsurface markers and functional characteristics. The hematopoietic stemcell, isolated from bone marrow, blood, cord blood, fetal liver and yolksac, is the progenitor cell that reinitiates hematopoiesis and generatesmultiple hematopoietic lineages. Hematopoietic stem cells can repopulatethe erythroid, neutrophil-macrophage, megakaryocyte and lymphoidhematopoietic cell pool. Stem cells that differentiate only to formcells of hematopoietic lineage, however, are unable to provide a sourceof cells for repair of other damaged tissues, for example, heart.

Neural stem cells were initially identified in the subventricular zoneand the olfactory bulb of fetal brain. Studies in rodents, non-humanprimates and humans, have shown that stem cells continue to be presentin adult brain. These stem cells can proliferate in vivo andcontinuously regenerate at least some neuronal cells in vivo. Whencultured ex vivo, neural stem cells can be induced to proliferate anddifferentiate into different types of neurons and glial cells. Whentransplanted into the brain, neural stem cells can engraft and generateneural cells and glial cells.

Mesenchymal stem cells (MSC), originally derived from the embryonalmesoderm and isolated from adult bone marrow, can differentiate to formmuscle, bone, cartilage, fat, marrow stroma, and tendon. Mesoderm alsodifferentiates into visceral mesoderm which can give rise to cardiacmuscle, smooth muscle, or blood islands consisting of endothelium andhematopoietic progenitor cells. All of the many mesenchymal stem cellsthat have been described have demonstrated limited differentiation tocells generally considered to be of mesenchymal origin. To date, thebest characterized mesenchymal stem cell reported is the cell isolatedby Pittenger, et al. (Science (1999) 284: 143-147 and U.S. Pat. No.5,827,740 (SH2⁺ SH4⁺ CD29⁺ CD44⁺ CD71⁺ CD90⁺ CD106⁺ CD120a ⁺ CD124⁺CD14⁻ CD34⁻ CD45⁻)). This cell is apparently limited in differentiationpotential to cells of the mesenchymal lineage.

There is a need, therefore, for a non-embryonic stem cell that has thecapacity to form differentiated cells of more than one embryoniclineage.

Myocardial Infarct (MI)

Myocardial infarction (MI) is characterized by the death of myocytes,coagulative necrosis, myocytolysis, contraction band necrosis, orapoptosis, resulting from a critical imbalance between the oxygen supplyand demand of the myocardium. The most common cause of MI is coronaryartery thrombosis following the rupture of atheromatous plaques. Thoughonce strictly defined as a lack of blood flow, the modern definition ofischemia emphasizes the imbalance between oxygen supply and demand aswell as the inadequate removal of metabolic waste products. Impairedoxygen delivery results in a reduction in oxidative phosphorylation thatcauses anaerobic glycolysis. This produces excess lactate thataccumulates in the myocardium. Impaired ATP production and acidosisresults in a decline in myocardial contractility. Similarly, ischemiareperfusion injury, without total occlusion, can also cause cardiacdamage.

The exposure of the contents of the plaque to the basement membranefollowing plaque rupture ultimately results in vessel blockageculminating from a series of events including platelet aggregation,thrombus formation, fibrin accumulation, and vasospasm. Total occlusionof the vessel for more than 4-6 hours results in irreversible myocardialnecrosis. Ultimately, death and morbidity from myocardial infarction isthe result of fatal dysrhythmia or progressive heart failure.Progressive heart failure is chiefly the result of insufficient musclemass (deficiency in muscle cells) or improper function of the heartmuscle, which can be caused by various conditions including, but notlimited to, hypertension. Progressive heart failure is, therefore, thefocus of cell-based therapy.

All current strategies for the treatment of myocardial infarction focuson limiting myocyte death. Annually in the United States, 500,000patients undergo angioplasty with stent placement. 400,000 will undergocoronary artery bypass, while an unknown additional number of patientswill be treated by thrombolytic therapy.

One approach, known as cellular cardiomyoplasty, has received recentattention and focuses on repopulation and engraftment of the injuredmyocardium by transplantation of healthy cells (Reffelmann, T. andKloner, R. A. (2003) Cardiovasc Res. 58(2): 358-68). Many cell typesthat might replace necrotic tissue and minimize regional scarring havebeen considered. Cells that have already committed to a specificlineage, such as satellite cells, cardiomyocytes, primary myocardialcell cultures, fibroblasts, and skeletal myoblasts, have been readilyused in cellular cardiomyoplasty with limited success in restoringdamaged tissue and improving cardiac function (Menasche, P. (2003)Cardiovasc Res. 58(2): 351-7; Etzion, S. et al (2001) J Mol CellCardiol. 33(7): 1321-30; Sakai, T. et al (1999) J. Thorac. Cardiovasc.Surg. 118(4): 715-24).

Cardiogenic progenitors are precursor cells that have committed to thecardiac lineage, but have not differentiated into cardiac muscle.Cardiomyocytes are the cells that comprise the heart. They are alsoknown as cardiac muscle cells. Use of cardiomyocytes in the repair ofcardiac tissue has been proposed. However, this approach is hindered byan inability to obtain sufficient quantities of cardiomyocytes for therepair of large areas of infarcted myocardium. Doubt has also been castover the incorporation and tissue-specific function of intra-cardiacgrafts derived from cardiomyocytes, even when they are harvested fromembryonic sources (Etzion, S. et al (2001) J. Mol. Cell. Cardiol. 33(7):1321-30). Intra-cardiac grafts using this cell type can be successfullygrafted and are able to survive in the myocardium after permanentcoronary artery occlusion and extensive infarction. However, engraftedrat embryonic cardiomyocytes attenuate, but do not fully reverse, leftventricular dilatation and prevent wall thinning. While survival wasimproved during 8 weeks of follow-up, the implanted cells did notdevelop into fully differentiated myocardium. Surprisingly, theyremained isolated from the host myocardium by scar tissue and did notimprove systolic function over time (Etzion, S. et al (2001) J. Mol.Cell. Cardiol. 33(7): 1321-30).

Congestive Heart Failure

Congestive heart failure (CHF) is a clinical condition in which aprimary or secondary circulatory system disease causes abnormal cardiacpressure or performance characteristics that lead to pulmonarycongestion (Zhang, J. and Narula, J. (2004) Surg Clin N Am 84:223-242).CHF is a chronic condition that results when the heart muscle is unableto pump blood as efficiently as is needed to maintain physiologicalhomeostasis. Although common causes of CHF include hypertension, anemiaand cardiomyopathy, CHF is most often caused by myocardial infarction(Lee, Michael, et al (2004) Reviews in Cardiovascular Medicine, 5:82-94). Currently, nearly 550,000 new cases of heart failure are nowdiagnosed each year.

CHF occurs when cardiac dysfunction prevents adequate perfusion ofperipheral tissues. Inadequate perfusion leads to stimulation ofcompensatory mechanisms that then cause many of the clinical signs andsymptoms of the condition. In patients with CHF, neurohumoralcompensatory mechanisms are activated. Chronic stimulation by theseagents increases cardiac afterload and preload, further worseningventricular function.

Small molecule therapeutics are being tested with the aim to disruptthese compensatory systems. However, no pharmacological interventionimproves the underlying pathophysiology of CHF.

Surgical intervention to treat CHF is also limited. Cardiactransplantation is the mainstay of treatment for patients with end-stagecardiomyopathies, such as CHF, but is limited by the scarcity of donororgans and complications, such as graft rejection and allograft coronaryvasculopathy (Fedak, P. et al (2003) Seminars in Thoracic andCardiovascular Surgery, 15: 277-286).

Recent studies challenge the traditional dogma that the heart is aterminally differentiated post-mitotic organ incapable of self-renewal.This opens the door to the possibility of delivering cells to treat CHF.It would be desirable to repopulate the injured cells with stem cells toregenerate cardiomyocytes and blood vessels, reverse ventricularremodeling, or reduce apoptosis of existing cells. Both myogenesis andangiogenesis may be required to restore cardiac function in patientswith transmural scar tissue. Thus, cells that are capable of inducingangiogenesis and forming muscle-like cells and endothelial cells may beuseful to reverse cardiac dysfunction in patients with CHF.

Clinical Experience with Stem Cells to Treat Cardiovascular Disease

Given the feasibility of the procedure, autologous skeletal muscle celltransplantation has been used clinically (Menasche P et al (2001) Lancet357:279-280). Autologous skeletal myoblasts were directly injected intononviable regions of the heart at the time of coronary bypass graftingor LVAD insertion. They formed viable grafts (Pagani F et al (2002)Circulation 106:11463 (abstract)). Patients reported improved symptomsand global heart functions. There was also evidence of viability in thedead region after clinical cell transplantation.

Both autologous bone marrow cells and myoblasts have been usedclinically in humans in a CHF setting. Strauer and co-workerstransplanted autologous mononuclear bone marrow cells in patients withacute myocardial infarction (Trauer et al (2002) Circulation 106:1913-1918). Regions of cell delivery demonstrated increased perfusion,viability, and wall motion, demonstrating improved myogenesis andangiogenesis.

Bone marrow cell-derived progenitor cells and circulating progenitorcells have also been tested (Assmus et al (2002) Circulation106:3009-3017). Treatment was associated with improved regional andglobal heart function, improved viability in the infarct area, andreduced left ventricular end-systolic volumes.

Human ES cells have been demonstrated to differentiate into myocyteswith many of the functional characteristics of cardiomyocytes (Cai, J.(2002) Neuromolecular Med, 2(3): 233-49). There are, however, safetyconcerns regarding therapeutic use because ES cells cause the formationof teratomas when administered to animals.

Adult stem cells that are dispersed throughout normal adults and can beisolated from a number of tissue sources, including organs, bone marrowand blood. They are free from many of the ethical and safety issuesassociated with ES cells (Cai, J. (2002) Neuromolecular Med, 2(3):233-49) and have not been linked to the growth of teratomas or canceroustumors. However, adult stem cells have shown limitations in theirpotential for therapeutic applications in that their differentiationpotential generally appears to be restricted to narrowly-defined celllineages or tissues, typically reflecting the tissue or organ from whichthe cells were isolated (Raff, M. (2003) Annu Rev Cell Dev Biol 19:1-22; Verfaillie, C. M. (2002) Trends Cell Biol, 12(11): 502-8).

Hematopoietic stem cells (HSC) have been utilized therapeutically forseveral decades in immune reconstitution settings.

In summary, loss of function and/or cell mass in cardiac muscle canarise, for example, by physical damage or disease-related damage (e.g.,genetic or acquired disease). Stem cell technology has made cellularmyoplasty a realistic treatment for restoring or enhancing cardiacmuscle function or cell mass. Tissue-specific stem cells and embryonicstem cells provide limited results.

SUMMARY OF THE INVENTION

A population of non-embryonic stem cells, designated by the inventors asmultipotent adult progenitor cells (MAPCs), can effectively restore orenhance cardiac muscle function in damaged tissues.

MAPC is an acronym for “multipotent adult progenitor cell” (non-ES,non-EG, non-germ) that has the capacity to differentiate into cells ofmore than one embryonic lineage. It can form cell types of all threeprimitive germ layers (ectoderm, mesoderm, and endoderm). The inventorswere the first to discover the existence of such a cell. Genes that arefound in ES cells were also found in the MAPC (e.g., telomerase, Oct3/4, rex-1, rox-1, sox-2). Thus the inventors identified a class of newmultipotent stem cells from non-embryonic and non-germ tissue.Telomerase or Oct 3/4 can be recognized as genes that are primaryproducts for the undifferentiated state. Telomerase, as discussedherein, is necessary for self-renewal. Oct 3/4 (Oct 3A in humans)appears to be specific for ES and germ cells.

Biologically and antigenically distinct from MSC, MAPC represents a moreprimitive progenitor cell population than MSC and demonstratesdifferentiation capability encompassing the epithelial, endothelial,neural, myogenic, hematopoeitic, osteogenic, hepatogenic, chondrogenicand adipogenic lineages (Verfaillie, C. M. (2002) Trends Cell Biol.12(11): 502-8, Jahagirdar, B. N., et al. (2001) Exp Hematol, 29(5):543-56). MAPCs thus represent a new class of non-embryonic stem cellthat emulates the broad biological plasticity characteristic of EScells, while maintaining the other characteristics that makenon-embryonic stem cells appealing. For example, MAPCs are capable ofindefinite culture without loss of their differentiation potential andshow efficient, long term, engraftment and differentiation alongmultiple developmental lineages in NOD-SCID mice without evidence ofteratoma formation (Reyes, M. and C. M. Verfaillie (2001) Ann N Y AcadSci, 938: 231-3).

MAPCs were initially isolated from bone marrow but subsequently havebeen established from other tissues, including brain, muscle, and cordblood (Jiang, Y., et al. (2002) Exp Hematol, 30(8): 896-904). Adherentcells from bone tissue are enriched in media containing low serum (2%),dexamethasone, EGF, PDGF, and other additives, and grown to highpopulation doublings. At early culture points more heterogeneity isdetected in the population. Then, many adherent stromal cells undergoreplicative senescence around cell doubling 30 and a more homogenouspopulation of cells continues to expand and maintains long telomeres.

The present invention, therefore, provides a method of repairing damagedheart tissue in a subject in need of such repair by contacting aneffective amount of multipotent adult stem cells with the damaged tissueof said subject. The cells can be autologous, allogenic or xenogenic. Inone embodiment the cells are allogeneic, so that cell banks areavailable for rapid response.

The invention further provides genetically engineered MAPCs toselectively express an endogenous gene or a transgene to treat tissuedamage.

MAPC or progeny can be used to treat cardiac diseases including, but notlimited to, myocarditis, cardiomyopathy, heart failure, damage caused byheart attacks, hypertension, atherosclerosis, and heart valvedysfunction. Progeny can include cardiomyocytes that repopulate theinjured tissue or endothelial cells that provide neo-vascularization tothe tissue.

The present invention relates to methods of improving function and/orcell mass in cardiac muscle tissue by administering MAPCs. MAPCs canengraft into the tissue to be treated in both healthy and damaged sites.

In another embodiment, the present invention comprises methods ofproviding cardiac muscle cells in a subject comprising administering anamount of MAPCs to the subject effective to generate new cardiac musclecells.

In another embodiment, the present invention comprises methods ofincreasing cardiac muscle mass in a subject comprising providing anamount of MAPCs to generate new cardiac muscle.

The methods can further comprise preliminary measures of isolating andexpanding a population of MAPCs.

In preferred embodiments, engraftment of MAPCs is in cardiac muscle inacute myocardial infarction.

In yet another embodiment, the present invention comprisesadministration of MAPCs to a subject in need thereof in the presence ofcytokines to enhance homing of MAPCs to damaged or abnormal cardiacmuscle, and to enhance differentiation of MAPCs or other progenitorsinto cells of the myogenic lineage (e.g., cardiomyocytes, myocytes,myoblasts and terminally-differentiated muscle cells).

In another aspect, the present invention comprises methods of increasingcardiac muscle in a subject comprising providing a suitable amount ofMAPCs to existing cardiac muscle and generating new cardiac muscle.

In yet another aspect, the present invention comprises methods ofincreasing cardiac muscle function in a subject comprising providingMAPCs to existing cardiac muscle in an amount sufficient to increasecardiac muscle function.

Cells integrating within, and therefore contributing to, the generationof new muscle can include endogenous stem or progenitor cells (e.g.,MAPCs), recruited stem or progenitor cells, exogenous MAPCs andcombinations thereof.

In one embodiment, the present invention comprises methods of improvingcardiac function in a subject comprising administering an amount ofMAPCs to the subject effective to improve cardiac function.

Evaluation of cardiac function can be monitored using various well-knownimaging techniques such as myocardial perfusion imaging, gated cardiacblood-pool imaging, first-pass ventriculography, right-to-left shuntdetection, positron emission tomography, single photon emission computedtomography, magnetic resonance imaging, harmonic phase magneticresonance imaging, echocardiography, and myocardial perfusion reserveimaging.

Administered MAPCs may contribute to generation of new tissue bydifferentiating into muscle cells in vivo. Alternatively, or inaddition, administered MAPCs may contribute to generation of new tissueby secreting cellular factors that aid in homing and recruitment ofendogenous MAPCs or other stem cells, such as cardiac, hematopoietic ormesenchymal stem cells, or other more differentiated cells, such asskeletal myoblasts, cardiac myoblasts and myocytes. Alternatively, or inaddition, MAPCs may secrete factors that act on endogenous stem orprogenitor cells in the target tissue causing them to differentiate inthe target site, thereby enhancing function. Further, MAPCs may secretefactors that act on stem, progenitor, or differentiated cells in thetarget tissue, causing them to divide. Further, MAPCs may provide forangiogenesis or reduce or prevent apoptosis.

In one embodiment, MAPC-based therapies can be used to treat damageresulting from disease states including congestive heart failure,coronary artery disease, myocardial infarction, myocardial ischemia,effects of atherosclerosis or hypertension, cardiomyopathy, cardiacarrhythmias, muscle degeneration, infective myocarditis, drug- andtoxin-induced muscle abnormalities, hypersensitivity myocarditis,autoimmune endocarditis, and congenital heart disease.

MAPCs can be administered to a subject by localized or systemicinjection. A variety of methods known in the art include, but are notlimited to, surgical intramyocardial injection, transendocardialinjection, intracoronary injection, transvascular injection,intramuscular injection, and intravenous injection. MAPCs may beadministered within or in proximity to the site requiring new musclecells, or enhanced function. Alternatively, they can be administered ata remote location.

The cells can be introduced into the body of the subject in conjunctionwith a suitable matrix implant. The matrix implant may provideadditional genetic material, cytokines, growth factors, or other factorsto promote growth and differentiation of the cells. The cells can beencapsulated prior to introduction into the body of the subject, such aswithin a polymer capsule.

Other aspects of the invention are described in or are obvious from thefollowing disclosure, and are within the ambit of the invention.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. Transcriptional profiling studies were performed to generategene and surface receptor-based markers that distinguish betweenmultipotent stem cells of the invention and more lineage-committed stemand progenitor cells. The experiments have resulted in a panel of 75markers having 10-fold different expression between MSC cultures and theMAPCs.

FIG. 2. Tri-lineage differentiation of GFP-labeled rat MAPC. Forendothelial differentiation, MAPCs were cultured on fibronectin-coatedplates in the presence of vascular endothelial growth factor (VEGF)-B.For hepatocyte differentiation, cells were grown on matrigel-coatedplates and treated with fibroblast growth factor-4 (FGF-4) andhepatocyte growth factor (HGF). Neuronal differentiation was induced bysequential treatment with basic-FGF (bFGF), with both FGF-8 and SonicHedgehog (SHH), and with brain-derived neurotrophic factor (BDNF). Aftertwo weeks mRNA was extracted from cells and applied to qPCR analysisusing primers specific for detection of various lineage markers. In allassays, cells cultured in the absence of lineage-inducing cytokinesserved as controls. The expression levels of lineage markers were firstnormalized to an internal control gene (GAPDH) which expression levelsare unaffected during differentiation. Differentiation success was thenassessed by calculation of the relative expression in the differentiatedor the control cells compared to the levels in the parental rat line,using an increase of more than 5-fold in the relative expression as acut-off for successful differentiation. Differentiated rat MAPCsdisplayed significant expression of the endothelial markers, vonWillebrand factor and PECAM-1 (top panel), the hepatic markers albumin,cytokeratin-18 and HNF-1a (middle panel), and the neuronal/astrocytemarkers GFAP, nestin and NF-200 (bottom panel).

FIG. 3. Schematic of the timeline following surgical induction ofmyocardial infarction and direct myocardiac injection of the MAPC. Thetimeline shows heart function analysis by echocardiography and the endpoint involving animal sacrifice to evaluate immune responses and stemcell engraftment. H&E: hematoxylin/eosin stain; α-GFP: detection byanti-GFP antibody.

FIG. 4. Assessment of the shortening fraction two weeks after MIinduction and MAPC injection. Lewis rats received either no MI surgery(no MI), or received LAD ligation followed by myocardial administrationof PBS (PBS) or 2 million MAPC in 5 separate injections of 400,000 cellsin the infarct border zone (MAPC). After two weeks animals weresubjected to echocardiography for measurement of the shorteningfraction. For each animal the left ventricular end diastolic dimension(LVEDD) and the left ventricular end systolic dimension (LVESD) weremeasured five separate times and the mean values were used to calculatethe shortening fraction (SF) according to the following formula:SF=(LVEDD-LVESD)/LVEDD*100. The asterisk in the figure indicates asignificant difference between the MAPC and the PBS control group(Dunn's test, nonparametric multiple comparisons).

FIG. 5. Morphological analysis of MI induction and MAPC engraftment. Twoweeks after MI surgery and MAPC injection (see FIG. 3), animals weresacrificed and the hearts were harvested. (A) Semi-thin cross-sectionsof plastic embedded tissue were stained with hematoxylin/eosin for grossmorphological evaluation. The infarcted region in the left ventricularwall is highlighted by the dotted line. (B, C and D) Sections werelabeled with primary goat anti-GFP antibody, followed by secondaryAlexa-fluor-488 conjugated anti-goat IgG antibody and DAPI stainingNuclei are blue and GFP positive cells are green. Autofluorescentsignals in the tissue are caused by lipofuscins (pink) and myofbrils(yellow). Orange signal in (D) is caused by erythrocytes. GFP positivecells are observed the infarcted heart tissue (A), in the border zone(B) and within areas containing healthy myofibrils. Note the extensionand re-arrangement of GFP positive MAPCs within the host cells in thetissue, and the apparent lack of accumulation of infiltratinginflammatory cells.

FIG. 6. Schematic of a phase II study showing long-term functionalbenefit, clearance and engraftment, and long-term immune response.

FIG. 7. Low immunogenicity (top panel) and immunosuppression (bottompanel) by MAPC as shown by mixed lymphocyte reaction assay (MLR). Top:B+B=donor B+donor B; B+A=donor B+donor A; B+K=donor B+donor K; B+R=donorB+donor R; B+T=donor B+donor T; B+BMPC=donor B+MAPC. The same result wasachieved with twelve different donors. Bottom: donor W+donor W; donorW+donor A; donor W+donor T; donor W+MSC; donor W+MAPC (17); donorW+donor A+MSC; donor W+donor A+MAPC (17); donor W+donor T+MSC; donorW+donor T+MAPC (17); donor W+donor P+MSC; donor W+donor P+MAPC (17).This figure shows that MAPC have immunosuppressive properties that arecharacteristic of MSC. They, therefore, provide the potential for invivo treatment with allogeneic MAPC.

DETAILED DESCRIPTION OF THE INVENTION

MAPC have the ability to regenerate all primitive germ layers(endodermal, mesodermal, and ectodermal) in vitro and in vivo. In thiscontext they are equivalent to embryonal stem cells, and distinct frommesenchymal stem cells, which are also isolated from bone marrow. Thebiological potency of these cells has been proven in various animalmodels, including mouse, rat, and xenogeneic engraftment of human stemcells in rats or NOD/SCID mice (Reyes, M. and C. M. Verfaillie (2001)Ann N Y Acad Sci. 938: 231-3; Jiang, Y. et al. (2002) Exp Hematol.30(8): 896-904). Clonal potency of this cell population has been shown.Single genetically marked MAPC were injected into mouse blastocysts,blastocysts implanted, and embryos developed to term (Jiang, Y. et al.(2002) Nature 418(6893): 41-9.). Post-natal analysis in highly chimericanimals showed reconstitution of all tissues and organs, includingliver. Dual staining experiments demonstrated that gene-marked stemcells contributed to a significant percentage of apparently functionalcardiomyocytes in these animals. These animals did not show any heartabnormalities or irregularities in either embryological or adult state.No abnormalities or organ dysfunction were observed in any of theseanimals.

Definitions

As used herein, the terms below are defined by the following meanings.

“MAPC” is an acronym for “multipotent adult progenitor cell”. It refersto a non-embryonic stem cell that can give rise to cell types of morethan one embryonic lineage. It can form cell lineages of all three germlayers (i.e., endoderm, mesoderm and ectoderm) upon differentiation.Like embryonic stem cells, human MAPCs express telomerase, Oct 3/4(i.e., Oct 3A), rex-1, rox-1 and sox-2 and telomerase. MAPC may expressSSEA-4, and nanog. The term “adult” in MAPC is non-restrictive. Itrefers to a non-embryonic somatic cell.

MAPCs constitutively express Oct 3/4 and high levels of telomerase(Jiang, Y. et al (2002) Nature 418:41-49; Exp Hematol. 30(8):896). MAPCsderived from human, mouse, rat or other mammals appear to be the onlynormal, non-malignant, somatic cell (i.e., non-germ cell) known to dateto express very high levels of telomerase even in late passage cells.The telomeres are extended in MAPCs and they are karyotypically normal.Because MAPCs injected into a mammal can migrate to and assimilatewithin multiple organs, MAPCs are self-renewing stem cells. As such,they have utility in the repopulation of organs, either in aself-renewing state or in a differentiated state compatible with theorgan of interest. They have the capacity to replace cell types thatcould have been damaged, died, or otherwise might have an abnormalfunction because of genetic or acquired disease. Or, as discussed below,they may contribute to the preservation of healthy cells or productionof new cells in a tissue.

“Multipotent”, with respect to MAPC, refers to the ability to give riseto cell types of more than one embryonic lineage. MAPC can form celllineages of all three primitive germ layers (i.e., endoderm, mesodermand ectoderm) upon differentiation.

“Expansion” refers to the propagation of a cell or cells withoutdifferentiation.

“Progenitor cells” are cells produced during differentiation of a stemcell that have some, but not all, of the characteristics of theirterminally-differentiated progeny. Defined progenitor cells, such as“cardiac progenitor cells,” are committed to a lineage, but not to aspecific or terminally-differentiated cell type. The term “progenitor”as used in the acronym “MAPC” does not limit these cells to a particularlineage.

The term “isolated” refers to a cell or cells not associated with one ormore cells or one or more cellular components that are associated withthe cell or cells in vivo. An “enriched population” means a relativeincrease in numbers of MAPC relative to one or more non-MAPC cell typesin vivo or in primary culture.

“Cardiomyocyte” refers to cells that comprise the heart and are alsoknown as cardiac muscle cells. A “myoblast” is a mononucleated,undifferentiated muscle precursor cell.

“Cardiogenic progenitors” are precursor cells that have committed to thecardiac lineage, but have not differentiated into cardiac muscle.

“Self-renewal” refers to the ability to produce replicate daughter stemcells having differentiation potential that is identical to those fromwhich they arose. A similar term used in this context is“proliferation.”

“Striated muscle” refers to muscle having actin and myosin filamentsaligned in orderly arrays to form a series of contractile units whichgive the cells a striated appearance. Types of striated muscle includeskeletal and cardiac muscle.

“Engraft” refers to the process of cellular contact and incorporationinto an existing tissue of interest in vivo.

“Persistence” refers to the ability of cells to resist rejection andremain and/or increase in number over time (e.g., days, weeks, months,years) in vivo.

The term “cytokines” means cellular factors that induce or enhancecellular movement, such as homing of MAPCs or other stem cells,progenitor cells or differentiated cells such as skeletal myoblasts,cardiac myoblasts, myocytes, and the like. Cytokines may also stimulatesuch cells to divide.

The phrase “differentiation factors” means cellular factors, preferablygrowth factors or angiogenic factors, that induce lineage commitment.

A “Subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, humans, farm animals,sport animals, and pets. Subjects in need of treatment by methods of thepresent invention include those suffering from a loss of function instriated muscle as a result of physical or disease-related damage.Disease states characterized by loss of striated muscle mass and/orfunction, and that benefit from methods of the present inventioninclude, but are not limited to, congestive heart failure, coronaryartery disease, myocardial infarction, myocardial ischemia,atherosclerosis, cardiomyopathy, cardiac arrhythmias, musculardystrophies, muscle mass abnormalities, muscle degeneration, myastheniagravis, infective myocarditis, drug- and toxin-induced muscleabnormalities, hypersensitivity myocarditis, autoimmune endocarditis,and congenital heart disease.

As used herein, “treat”, “treating”, or “treatment” includes treating,preventing, ameliorating, inhibiting or removing an injury ordisease-related condition and/or symptom of an injury or disease-relatedcondition.

An “effective amount” generally means an amount which provides thedesired local or systemic effect, such as enhanced performance. Forexample, an effective dose is an amount sufficient to effect abeneficial or desired clinical result. The dose can be administered inone or more administrations and can include any preselected amount ofcells. The precise determination of what would be considered aneffective dose may be based on factors individual to each subject,including size, age, injury and/or disease or injury being treated andamount of time since the injury occurred or the disease began. Oneskilled in the art, particularly a physician, would be able to determinethe number of cells that would constitute an effective dose.

“Co-administer” can include simultaneous or sequential administration oftwo or more agents.

Administered MAPCs may contribute to generation of new tissue bydifferentiating into specific cells, such as cardiomyocytes, in vivo.Alternatively, or in addition, administered MAPCs may contribute togeneration of new tissue by secreting cellular factors that aid inhoming and recruitment of endogenous MAPCs or other stem cells, or othermore differentiated cells, such as cardiomyocytes. Alternatively, or inaddition, MAPCs may secrete factors that act on endogenous stem orprogenitor cells in the target tissue causing them to differentiate inthe target site, thereby enhancing function. Further, MAPCs may secretefactors that act on stem, progenitor, or differentiated cells in thetarget tissue, causing them to divide. Thus, MAPCs may provide benefitthrough trophic influences. Examples of trophic influences include, butare not limited to, limiting inflammatory damage, limiting vascularpermeability, improving cell survival and homing of repair cells tosites of damage. Additionally, MAPCs may also provide benefit byincreasing capillary density and stimulating angiogenesis. This may beachieved by production of angiogenic factors, such as VEGF, or bydifferentiation of the MAPCs and inclusion in new vessel tissue, orboth. Therapeutic benefit may be achieved by a combination of the abovepathways.

The terms “comprises”, “comprising”, and the like can have the meaningascribed to them in U.S. Patent Law and can mean “includes”, “including”and the like. As used herein, “including” or “includes” or the likemeans including, without limitation.

Administration of MAPCs

MAPCs can be administered to a subject by a variety of methods known inthe art. Preferably, administration is through injection, including butnot limited to, surgical intramyocardial injection, transendocardialinjection, intracoronary injection, transvascular injection,intramuscular injection, and intravenous injection.

Surgical myocardial injection is an invasive approach that may beparticularly suitable to patients who are undergoing concurrent surgicalprocedures. The injection process can be performed under directvisualization, such as by using a small gauge needle, thereby allowingevaluation by direct inspection of potential target zones. Not all areasof the myocardium can be readily accessed with this approach. Usually,multiple injections are made in a particular region, usually around aborder zone. Direct injection can be performed when the patient isconnected to a heart lung machine, in which case the cells can be veryprecisely administered.

Transendocardial injection primarily involves a catheter system known asNOGA. The injection catheter incorporates the mapping capabilities ofthe system. This provides a means by which tissues with differentdegrees of viability and ischemia can be mapped in detail, allowingtherapy to be precisely targeted (e.g., at the border zone of aninfarct). NOGA catheters can be used alongside detection techniques suchas magnetic resonance imaging (MRI), echocardiogram (EKG), or computedtomographic techniques such as positron emission tomography (PET) orsingle photon emission computed tomography (SPECT).

Intracoronary injection is particularly well suited for delivery ofcells to a specific coronary territory. It is less complex thantransendocardial delivery, and because of the segmental nature ofcoronary artery disease, may be more practical than other techniques.Where retention of cells in the target area is at issue, this techniqueis especially suited for treating relatively intense ischemia. Thequantity of cells and time of infusion can be carefully monitored toavoid coronary flow impairment and myocardial cell necrosis.

Intravenous injection is the simplest method of cell administration,although a greater degree of dependence on homing of the stem cells isrequired for them to reach the tissue of interest (e.g., myocardium).Carefully controlled dosing, which is readily determined by one skilledin the art, enhances this method of administration.

With respect to timing of administration for myocardial infarction, thepreferred embodiment would likely be a few days after the event or atleast after the patient is sufficiently stabilized. Current clinicaltrials in the art administer cell therapy about four days after theevent (see, for example, Circulation (2002)106:3009-3017).

For congestive heart failure the preferred embodiment would betransendocardial via catheter or direct injection. Also, for congestiveheart failure, preferred embodiments encompass bioengineered patches ofcells contained within biodegradable matrices. For coronary arterydisease the preferred embodiment would be intracoronary in order tostimulate neovascularization and also to potentially treat underlyingatherosclerosis.

Certain cytokines can alter or affect the migration of MAPCs or theirdifferentiated counterparts to the site of damaged muscle tissue.“Homing” of stem cells to the injured muscle tissues would concentratethe implanted cells in an environment favorable to their growth andfunction. In more acutely ischemic situations, the stem cells can beadministered either peripherally or locally through the circulatorysystem. When the homing signals may be less intense, as may be the casefor chronic ischemic or nonischemic cardiomyopathies, injection of thecells directly into the cardiac muscle may produce a more favorableoutcome. Pre-treatment of a patient with cytokine(s) to promote homingis another alternative contemplated in the methods of the presentinvention.

Cytokines include, but are not limited to, stromal cell derived factor-1(SDF-1), stem cell factor (SCF), angiopoietin-1, placenta-derived growthfactor (PIGF) and granulocyte-colony stimulating factor (G-CSF).Cytokines also include any which promote the expression of endothelialadhesion molecules, such as ICAMs, VCAMs, and others, which facilitatethe homing process.

Differentiation of MAPCs to a phenotype characteristic of musculartissues can be enhanced when differentiation factors are employed.Differentiation factors promoting muscle formation include, but are notlimited to vascular endothelial growth factor (VEGF), fibroblast growthfactors (e.g., FGF4, FGF8, bFGF) Wnt11, DKK1, ascorbic acid,isoproterenol and endothelin.

Viability of newly forming tissues can be enhanced by angiogenesis.Differentiation factors promoting angiogenesis include, but are notlimited to, VEGF, aFGF, angiogenin, angiotensin-1 and -2, betacellulin,bFGF, Factor X and Xa, HB-EGF, PDGF, angiomodulin, angiotropin,angiopoetin-1, prostaglandin E1 and E2, steroids, heparin,1-butyryl-glycerol and nicotinic amide.

Factors that decrease apoptosis can also promote the formation of newstriated muscle. Factors that decrease apoptosis include, but are notlimited to, β-blockers, angiotensin-converting enzyme inhibitors (ACEinhibitors), AKT, HIF, carvedilol, angiotensin II type 1 receptorantagonists, caspase inhibitors, cariporide, and eniporide.

Exogenous factors (e.g., cytokines, differentiation factors andanti-apoptosis factors) can be administered prior to, after orconcomitantly with MAPCs. For example, a form of concomitantadministration would comprise combining a factor of interest in the MAPCsuspension media prior to administration. Administrations are variableand may include an initial administration followed by subsequentadministrations, ascertained by the skilled artisan from thisdisclosure, and the knowledge in the art.

A method to potentially increase cell survival is to incorporate MAPCsor other cells of interest into a biopolymer or synthetic polymer.Depending on the patient's condition, the site of injection might proveinhospitable for cell seeding and growth because of scarring or otherimpediments. Examples of biopolymer include, but are not limited to,cells mixed with fibronectin, fibin, fibrinogen, thrombin, collagen, andproteoglycans. This could be constructed with or without includedcytokines or differentiation factors. Additionally, these could be insuspension but residence time at sites subjected to flow would benominal. Another alternative is a three-dimensional gel with cellsentrapped within the interstices of the cell biopolymer admixture. Againdifferentiation factors or cytokines could be included within the cells.These could be deployed by injection via various routes describedherein, via cardiac catheters or other surgical procedures.

In current human studies of autologous mononuclear bone marrow cells,empirical doses ranging from 1 to 4×10⁷ cells have been used. However,different scenarios may require optimization of the amount of cellsinjected into a tissue of interest. Thus, the quantity of cells to beadministered will vary for the subject being treated. In a preferredembodiment, between 10⁴ to 10⁸, more preferably 10⁵ to 10⁷, and mostpreferably, 3×10⁷ cells and optionally, 50 to 500 μg/kg per day of acytokine can be administered to a human subject. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including their size, age, sizeof the infarct or other muscular damage, and amount of time since thedamage occurred and factors associated with the mode of delivery (directinjection—lower doses, intravenous—higher doses). Therefore, dosages canbe readily ascertained by those skilled in the art from this disclosureand the knowledge in the art.

Another issue regarding the use of stem cells is the purity of theisolated stem cell population. Bone marrow cells, for example, comprisemixed populations of cells, which can be purified to a degree sufficientto produce a desired effect. Those skilled in the art can readilydetermine the percentage of MAPCs in a population using variouswell-known methods, such as fluorescence activated cell sorting (FACS).Preferable ranges of purity in populations comprising MAPCs are about50-55%, 55-60%, and 65-70%. More preferably the purity is about 70-75%,75-80%, 80-85%; and most preferably the purity is about 85-90%, 90-95%,and 95-100%. However, populations with lower purity can also be useful,such as about 25-30%, 30-35%, 35-40%, 40-45% and 45-50%. Purity of MAPCscan be determined according to the gene expression profile within apopulation. Dosages can be readily adjusted by those skilled in the art(e.g., a decrease in purity may require an increase in dosage).

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active stem cell(s) and/or cytokine(s)) are present inan amount of 0.001 to 50 wt % solution in phosphate buffered saline, andthe active ingredient is present in the order of micrograms tomilligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt %or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %,and most preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD₅₀ in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

When administering a therapeutic composition of the present invention,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions anddispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyol (for example, glycerol, propylene glycol, liquid polyethyleneglycol, and the like) and suitable mixtures thereof.

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the cells.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired.

In one embodiment, MAPCs can be administered initially, and thereaftermaintained by further administration of MAPCs. For instance, MAPCs canbe administered by one method of injection, and thereafter furtheradministered by a different or the same type of method. For example,MAPCs can be administered by surgical myocardial injection to bringcardiovascular function to a suitable level. The patient's levels canthen maintained, for example, by intravenous injection, although otherforms of administration, dependent upon the patient's condition, can beused.

It is noted that human subjects are treated generally longer than thecanines or other experimental animals, such that treatment has a lengthproportional to the length of the disease process and effectiveness. Thedoses may be single doses or multiple doses over a period of severaldays. Thus, one of skill in the art can scale up from animalexperiments, e.g., rats, mice, canines and the like, to humans, bytechniques from this disclosure and documents cited herein and theknowledge in the art, without undue experimentation. The treatmentgenerally has a length proportional to the length of the disease processand drug effectiveness and the subject being treated.

Examples of compositions comprising MAPCs include liquid preparationsfor administration, including suspensions; and, preparations forintramuscular or intravenous administration (e.g., injectableadministration), such as sterile suspensions or emulsions. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can also be lyophilized. Thecompositions can contain auxiliary substances such as wetting oremulsifying agents, pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation.

Compositions of the invention are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions, which may be buffered to a selected pH. Liquidpreparations are normally easier to prepare than gels, other viscouscompositions, and solid compositions. Additionally, liquid compositionsare somewhat more convenient to administer, especially by injection.Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form, such as a time release form or liquid-filled form).

Solutions, suspensions and gels normally contain a major amount of water(preferably purified, sterilized water) in addition to the cells. Minoramounts of other ingredients such as pH adjusters (e.g., a base such asNaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents and jelling agents (e.g.,methylcellulose), may also be present. The compositions can be isotonic,i.e., they can have the same osmotic pressure as blood and lacrimalfluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount, which will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative or cell stabilizer can beemployed to increase the life of the compositions. Preferably, ifpreservatives are necessary, it is well within the purview of theskilled artisan to select compositions that will not affect theviability or efficacy of the MAPCs as described in the presentinvention.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert. This willpresent no problem to those skilled in chemical and pharmaceuticalprinciples, or problems can be readily avoided by reference to standardtexts or by simple experiments (not involving undue experimentation),from this disclosure and the documents cited herein.

Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the composition form used for administration(e.g., solid vs. liquid). Dosages for humans or other mammals can bedetermined without undue experimentation by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

Suitable regimes for initial administration and further doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,can be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, and the knowledge in the art.

MAPCs

Human MAPCs are described in U.S. patent application Ser. Nos.10/048,757 (PCT/US00/21387 (published as WO 01/11011)) and Ser. No.10/467,963 (PCT/US02/04652 (published as WO 02/064748)), the contents ofwhich are incorporated herein by reference for their description ofMAPCs. MAPCs have been identified in other mammals. Murine MAPCs, forexample, are also described in PCT/US00/21387 (published as WO 01/11011)and PCT/US02/04652 (published as WO 02/064748). Rat MAPCs are alsodescribed in WO 02/064748.

Isolation and Growth of MAPCs

Methods of MAPC isolation for humans and mouse are known in the art.They are described in PCT/US00/21387 (published as WO 01/11011) and forrat in PCT/US02/04652 (published as WO 02/064748), and these methods,along with the characterization of MAPCs disclosed therein, areincorporated herein by reference.

MAPCs were initially isolated from bone marrow, but were subsequentlyestablished from other tissues, including brain and muscle (Jiang Y, etal., Nature. 2002; 418:41-49; Jiang Y, et al., Exp Hematol. 2002b;30:896-904). Thus, MAPCs can be isolated from multiple sources,including bone marrow, placenta, umbilical cord and cord blood, muscle,brain, liver, spinal cord, blood or skin. For example, MAPCs can bederived from bone marrow aspirates, which can be obtained by standardmeans available to those of skill in the art (see, for example,Muschler, G. F., et al. J. Bone Joint Surg. Am. 1997; 79(11):1699-709;Batinic, D., et al., Bone Marrow Transplant. 1990; 6(2):103-7) It is,therefore, now possible for one of skill in the art to obtain bonemarrow aspirates, brain or liver biopsies, and other organs, and isolatethe cells using positive or negative selection techniques available tothose of skill in the art, relying upon the genes that are expressed (ornot expressed) in these cells (e.g., by functional or morphologicalassays, such as those disclosed in the above-referenced applications,which have been incorporated herein by reference).

MAPCs from Human Bone Marrow as Described in PCT/US00/21387

Bone marrow mononuclear cells were derived from bone marrow aspirates,which were obtained by standard means available to those of skill in theart (see, for example, Muschler, G. F., et al. J. Bone Joint Surg. Am.1997; 79(11):1699-709; Batinic, D., et al., Bone Marrow Transplant.1990; 6(2):103-7). Multipotent adult stem cells are present within thebone marrow (or other organs such as liver or brain), but do not expressthe common leukocyte antigen CD45 or erythroblast specific glycophorin-A(Gly-A). The mixed population of cells was subjected to a Ficoll Hypaqueseparation. The cells were then subjected to negative selection usinganti-CD45 and anti-Gly-A antibodies, depleting the population of CD45⁺and Gly-A⁺ cells, and the remaining approximately 0.1% of marrowmononuclear cells were then recovered. Cells could also be plated infibronectin-coated wells and cultured as described below for 2-4 weeksto deplete the population of cells of CD45⁺ and Gly-A⁺ cells.

Alternatively, positive selection could be used to isolate cells via acombination of cell-specific markers. Both positive and negativeselection techniques are available to those of skill in the art, andnumerous monoclonal and polyclonal antibodies suitable for negativeselection purposes are also available in the art (see, for example,Leukocyte Typing V, Schlossman, et al., Eds. (1995) Oxford UniversityPress) and are commercially available from a number of sources.

Techniques for mammalian cell separation from a mixture of cellpopulations have also been described by Schwartz, et al., in U.S. Pat.No. 5,759,793 (magnetic separation), Basch, et. al., J. Immunol.Methods. 1983; 56:269 (immunoaffinity chromatography), and Wysocki andSato, Proc. Natl. Acad. Sci. (USA) 1978; 75:2844 (fluorescence-activatedcell sorting).

Recovered CD45⁻/GlyA⁻ cells were plated onto culture dishes coated with5-115 ng/ml (about 7-10 ng/ml can be used) serum fibronectin or otherappropriate matrix coating. Cells were maintained in Dulbecco's MinimalEssential Medium (DMEM) or other appropriate cell culture medium,supplemented with 1-50 ng/ml (about 5-15 ng/ml can be used)platelet-derived growth factor-BB (PDGF-BB), 1-50 ng/ml (about 5-15ng/ml can be used) epidermal growth factor (EGF), 1-50 ng/ml (about 5-15ng/ml can be used) insulin-like growth factor (IGF), or 100-10,000 IU(about 1,000 IU can be used) LIF, with 10⁻¹⁰ to 10⁻⁸ M dexamethasone (orother appropriate steroid), 2-10 μg/ml linoleic acid, and 0.05-0.15 μMascorbic acid. Other appropriate media include, for example, MCDB, MEM,IMDM, and RPMI. Cells can either be maintained without serum, in thepresence of 1-2% fetal calf serum, or, for example, in 1-2% human ABserum or autologous serum.

When re-seeded at 2×10³ cells/cm² every 3 days, >40 cell doublings wereroutinely obtained, and some populations underwent >70 cell doublings.Cell doubling time was 36-48 h for the initial 20-30 cell doublings.Afterwards cell-doubling time was extended to as much as 60-72 h.

Telomere length of MAPCs from 5 donors (age about 2 years to about 55years) cultured at re-seeding densities of 2×10³ cells/cm² for 23-26cell doublings was between 11-13 KB. This was 3-5 KB longer thantelomere length of blood lymphocytes obtained from the same donors.Telomere length of cells from 2 donors evaluated after 23 and 25 celldoublings, respectively, and again after 35 cells doublings, wasunchanged. The karyotype of these MAPCS was normal.

Phenotype of Human MAPCs Under Conditions Described in PCT/US00/21387

Immunophenotypic analysis by FACS of human MAPCs obtained after 22-25cell doublings indicated that the cells do not express CD31, CD34, CD36,CD38, CD45, LD50, CD62E and -P, HLA-DR, Muc18, STRO-1, cKit, Tie/Tek;express low levels of CD44, HLA-class I, and β2-microglobulin, expressCD10, CD13, CD49b, CD49e, CDw90, Flk1 (N>10).

Once cells underwent >40 doublings in cultures re-seeded at about2×10³/cm², the phenotype became more homogenous and no cell expressedHLA class-I or CD44 (n=6). When cells were grown at higher confluence,they expressed high levels of Muc18, CD44, HLA class 1 andβ2-microglobulin, which is similar to the phenotype described for MSC(N=8) (Pittenger et al. Science 1999; 284:143-147).

Immunhistochemistry showed that human MAPCs grown at about 2×10³/cm²seeding density expressed EGF-R, TGF-R1 and -2, BMP-R1A, PDGF-R1a and-B, and that a small subpopulation (between 1 and 10%) of MAPCs stainedwith anti-SSEA4 antibodies (Kannagi, R. EMBO J 1983; 2:2355-61).

Using Clontech cDNA arrays the expressed gene profile of human MAPCscultured at seeding densities of about 2×10³ cells/cm² for 22 and 26cell doublings was determined:

-   -   A. MAPCs did not express CD31, CD36, CD62E, CD62P, CD44-H, cKit,        Tie, receptors for IL1, IL3, IL6, IL11, G CSF, GM-CSF, Epo,        Flt3-L, or CNTF, and low levels of HLA-class-I, CD44-E and        Muc-18 mRNA.    -   B. MAPCs expressed mRNA for the cytokines BMP1, BMP5, VEGF, HGF,        KGF, MCP1; the cytokine receptors Flk1, EGF-R, PDGF-R1α, gp130,        LIF-R, activin-R1 and -R2, TGFR-2, BMP-R1A; the adhesion        receptors CD49c, CD49d, CD29; and CD10.    -   C. MAPCs expressed mRNA for hTRT and TRF1; the POU domain        transcription factor Oct-4, sox-2, sox 11 (neural development),        sox 9 (chondrogenesis) (Lefebvre V. Matrix Biol 1988;        16:529-40); homeodeomain transcription factors: Hoxa4 and -a5        (cervical and thoracic skeleton specification; organogenesis of        respiratory tract) (Packer A. I. Dev Dyn 2000; 17:62-74), Hox-a9        (myelopoiesis) (Lawrence H. Blood 1997; 89:1922), Dlx4        (specification of forebrain and peripheral structures of head)        (Akimenko M. A. J Neurosci 1994; 14:3475-86), MSX1 (embryonic        mesoderm, adult heart and muscle, chondro- and osteogenesis)        (Foerst-Potts L. Dev Dyn 1997; 209:70-84), PDX1 (pancreas)        (Offield M. F. Development 1996; 122:983-95).    -   D. Presence of Oct-4, LIF-R, and hTRT mRNA was confirmed by        RT-PCR.    -   E. In addition, RT-PCR showed that Rex-1 mRNA and Rox-1 mRNA        were expressed in MAPCs.

Oct-4, Rex-1 and Rox-1 were expressed in MAPCs derived from human andmurine marrow and from murine liver and brain. Human MAPCs expressedLIF-R and stained positive with SSEA-4. Finally, Oct-4, LIF-R, Rex-1 andRox-1 mRNA levels were found to increase in human MAPCs cultured beyond30 cell doublings, which resulted in phenotypically more homogenouscells. In contrast, MAPCs cultured at high density lost expression ofthese markers. This was associated with senescence before 40 celldoublings and loss of differentiation to cells other than chondroblasts,osteoblasts and adipocytes.

Culturing MAPCs as Described in PCT/US00/21387

MAPCs isolated as described herein can be cultured using methodsdisclosed herein and in PCT/US00/21387, which is incorporated byreference for these methods.

Briefly, for the culture of MAPCs, culture in low-serum or serum-freemedium was preferred to maintain the cells in the undifferentiatedstate. Serum-free medium used to culture the cells, as described herein,was supplemented as described in Table 1. Human MAPCs do not requireLIF.

TABLE 1 Insulin 10-50 μg/ml (10 μg/ml)* Transferrin 0-10 μg/ml (5.5μg/ml) Selenium 2-10 ng/ml (5 ng/ml) Bovine serum albumin (BSA) 0.1-5μg/ml (0.5 μg/ml) Linoleic acid 2-10 μ/ml (4.7 μg/ml) Dexamethasone0.005-0.15 μM (.01 μM) L-ascorbic acid 2-phosphate 0.1 mM Low-glucoseDMEM (DMEM-LG) 40-60% (60%) MCDB-201 40-60% (40%) Fetal calf serum 0-2%Platelet-derived growth 5-15 ng/ml (10 ng/ml) Epidermal growth factor5-15 ng/ml (10 ng/ml) Insulin like growth factor 5-15 ng/ml (10 ng/ml)Leukemia inhibitory factor 10-10,000 IU (1,000 IU) *Preferredconcentrations are shown in parentheses.

Addition of 10 ng/mL LIF to human MAPCs did not affect short-term cellgrowth (same cell doubling time till 25 cell doublings, level of Oct-4(Oct 3/4) expression). In contrast to what was seen with human cells,when fresh murine marrow mononuclear cells, depleted on day 0 of CD45⁺cells, were plated in MAPC culture, no growth was seen. When murinemarrow mononuclear cells were plated, and cultured cells 14 days laterdepleted of CD45⁺ cells, cells with the morphology and phenotype similarto that of human MAPCs appeared. This suggested that factors secreted byhemopoietic cells were needed to support initial growth of murine MAPCs.When cultured with PDGF-BB and EFG alone, cell doubling was slow (>6days) and cultures could not be maintained beyond 10 cell doublings.Addition of 10 ng/ml LIF significantly enhanced cell growth.

Once established in culture, cells could be frozen and stored as frozenstocks, using DMEM with 40% FCS and 10% DMSO. Other methods forpreparing frozen stocks for cultured cells are also available to thoseof skill in the art.

Thus, MAPCs could be maintained and expanded in culture medium that isavailable to the art. Such media include, but are not limited toDulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 Medium®, Eagle'sMinimum Essential Medium®, F-12K Medium®, Iscove's Modified Dulbecco'sMedium® and RPMI-1640 Medium®. Many media are also available as alow-glucose formulations, with or without sodium pyruvate.

Also contemplated is supplementation of cell culture medium withmammalian sera. Sera often contain cellular factors and components thatare necessary for viability and expansion. Examples of sera includefetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calfserum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum(HS), human serum, chicken serum, porcine serum, sheep serum, rabbitserum, serum replacements, and bovine embryonic fluid. It is understoodthat sera can be heat-inactivated at 55-65° C. if deemed necessary toinactivate components of the complement cascade.

Additional supplements can also be used advantageously to supply thecells with the necessary trace elements for optimal growth andexpansion. Such supplements include insulin, transferrin, sodiumselenium and combinations thereof. These components can be included in asalt solution such as, but not limited to Hanks' Balanced Salt Solution®(HBSS), Earle's Salt Solution®, antioxidant supplements, MCDB -201®supplements, phosphate buffered saline (PBS), ascorbic acid and ascorbicacid-2-phosphate, as well as additional amino acids. Many cell culturemedia already contain amino acids, however some require supplementationprior to culturing cells. Such amino acids include, but are not limitedto, L-alanine, L-arginine, L-aspartic acid, L-asparagine, L-cysteine,L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine,L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine,L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, andL-valine. It is well within the skill of one in the art to determine theproper concentrations of these supplements.

Antibiotics are also typically used in cell culture to mitigatebacterial, mycoplasmal, and fungal contamination. Typically, antibioticsor anti-mycotic compounds used are mixtures of penicillin/streptomycin,but can also include, but are not limited to amphotericin (Fungizone®),ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin,mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin,polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin,and zeocin. Antibiotic and anti-mycotic additives can be of someconcern, depending on the type of work being performed. One possiblesituation that can arise is an antibiotic-containing media whereinbacteria are still present in the culture, but the action of theantibiotic performs a bacteriostatic rather than bacteriocidalmechanism. Also, antibiotics can interfere with the metabolism of somecell types.

Hormones can also be advantageously used in cell culture and include,but are not limited to D-aldosterone, diethylstilbestrol (DES),dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin,progesterone, somatostatin/human growth hormone (HGH), thyrotropin,thyroxine, and L-thyronine.

Lipids and lipid carriers can also be used to supplement cell culturemedia, depending on the type of cell and the fate of the differentiatedcell. Such lipids and carriers can include, but are not limited tocyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated toalbumin, linoleic acid and oleic acid conjugated to albumin,unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugatedto albumin, oleic acid unconjugated and conjugated to albumin, amongothers.

Also contemplated is the use of feeder cell layers. Feeder cells areused to support the growth of fastidious cultured cells, particularly EScells. Feeder cells are normal cells that have been inactivated byγ-irradiation. In culture, the feeder layer serves as a basal layer forother cells and supplies cellular factors without further growth ordivision of their own (Lim, J. W. and Bodnar, A., Proteomics 2002;2(9):1187-1203 (2002)). Examples of feeder layer cells are typicallyhuman diploid lung cells, mouse embryonic fibroblasts, Swiss mouseembryonic fibroblasts, but can be any post-mitotic cell that is capableof supplying cellular components and factors that are advantageous inallowing optimal growth, viability, and expansion of stem cells. In manycases, feeder cell layers are not necessary to keep the ES cells in anundifferentiated, proliferative state, as leukemia inhibitory factor(LIF) has anti-differentiation properties. Therefore, supplementationwith LIF could be used to maintain MAPC in some species in anundifferentiated state.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components and synthetic orbiopolymers. Stem cells often require additional factors that encouragetheir attachment to a solid support, such as type I, type II, and typeIV collagen, concanavalin A, chondroitin sulfate, fibronectin,“superfibronectin” and fibronectin-like polymers, gelatin, laminin,poly-D and poly-L-lysine, thrombospondin, and vitronectin.

The maintenance conditions of stem cells can also contain cellularfactors that allow stem cells, such as MAPCs, to remain in anundifferentiated form. It is advantageous under conditions where thecell must remain in an undifferentiated state of self-renewal for themedium to contain epidermal growth factor (EGF), platelet derived growthfactor (PDGF), leukemia inhibitory factor (LIF; in selected species),and combinations thereof. It is apparent to those skilled in the artthat supplements that allow the cell to self-renew but not differentiateshould be removed from the culture medium prior to differentiation.

Stem cell lines and other cells can benefit from co-culturing withanother cell type. Such co-culturing methods arise from the observationthat certain cells can supply yet-unidentified cellular factors thatallow the stem cell to differentiate into a specific lineage or celltype. These cellular factors can also induce expression of cell-surfacereceptors, some of which can be readily identified by monoclonalantibodies. Generally, cells for co-culturing are selected based on thetype of lineage one skilled in the art wishes to induce, and it iswithin the capabilities of the skilled artisan to select the appropriatecells for co-culture.

Methods of identifying and subsequently separating differentiated cellsfrom their undifferentiated counterparts can be carried out by methodswell known in the art. Cells that have been induced to differentiate canbe identified by selectively culturing cells under conditions wherebydifferentiated cells outnumber undifferentiated cells. Similarly,differentiated cells can be identified by morphological changes andcharacteristics that are not present on their undifferentiatedcounterparts, such as cell size, the number of cellular processes (i.e.formation of dendrites and/or branches), and the complexity ofintracellular organelle distribution. Also contemplated are methods ofidentifying differentiated cells by their expression of specificcell-surface markers such as cellular receptors and transmembraneproteins. Monoclonal antibodies against these cell-surface markers canbe used to identify differentiated cells. Detection of these cells canbe achieved through fluorescence activated cell sorting (FACS), andenzyme-linked immunosorbent assay (ELISA). From the standpoint oftranscriptional upregulation of specific genes, differentiated cellsoften display levels of gene expression that are different fromundifferentiated cells. Reverse-transcription polymerase chain reaction(RT-PCR) can also be used to monitor changes in gene expression inresponse to differentiation. In addition, whole genome analysis usingmicroarray technology can be used to identify differentiated cells.

Accordingly, once differentiated cells are identified, they can beseparated from their undifferentiated counterparts, if necessary. Themethods of identification detailed above also provide methods ofseparation, such as FACS, preferential cell culture methods, ELISA,magnetic beads, and combinations thereof. A preferred embodiment of theinvention envisions the use of FACS to identify and separate cells basedon cell-surface antigen expression.

Additional Culture Methods

In additional experiments it has been found that the density at whichMAPCs are cultured can vary from about 100 cells/cm² or about 150cells/cm² to about 10,000 cells/cm², including about 200 cells/cm² toabout 1500 cells/cm² to about 2000 cells/cm². The density can varybetween species. Additionally, optimal density can vary depending onculture conditions and source of cells. It is within the skill of theordinary artisan to determine the optimal density for a given set ofculture conditions and cells.

Also, effective atmospheric oxygen concentrations of less than about10%, including about 3-5%, can be used at any time during the isolation,growth and differentiation of MAPCs in culture.

Inducing MAPCs to Differentiate to Form Committed Progenitors andTissue-Specific Cell Types

Using appropriate growth factors, chemokines, and cytokines, MAPCs canbe induced to differentiate to form a number of cell lineages,including, for example, a variety of cells of mesodermal origin as wellas cell from neuroectodermal origin (glial cells, oligodendrocytes, andneurons) as well as endodermal origin. These methods are known in theart and are described in both U.S. and PCT applications mentioned above,incorporated by reference for disclosing these methods.

Cardiac muscle differentiation can be accomplished by adding about 5 toabout 200 ng/ml (preferably about 100 ng/ml) basic fibroblast growthfactor (bFGF) to the standard serum-free culture media without growthfactors, as previously described. Confluent MAPCs were exposed to about2 to 5 μM (preferably about 3 μM) 5-azacytidine and to about 10⁻⁵-10⁻⁷ M(preferably about 10⁻⁶ M) retinoic acid, and then cultured in MAPCexpansion medium afterwards. Alternatively, MAPCs were cultured witheither of these inducers alone or a combination of both and thencultured in serum-free medium with about 50-200 ng/mL (preferably about100 ng/mL FGF2 or a combination of about 5-20 ng/mL (preferably about 10ng/mL) BMP-4 and about 100 ng/mL FGF2. Expression of proteins consistentwith cardiomyocytes was found. Gata4 and Gata6 were expressed as earlyas day 2 and persisted till day 15. Cardiac troponin-T was expressedafter day 4 and cardiac troponin-I from day 6 on, while we could detectANP after day 11. These cardiac proteins were detected in >70% of cellsby immunohistochemistry on day 15. The transcription factor Myf6 wasfound from day 2 on. Expression of desmin started on day 6 and myogeninon day 2. Skeletal actin was also found. When the cultures weremaintained for >3 weeks, cells formed syncitia. There also wereinfrequent spontaneous contractions occurring in the cultures, whichwere propagated over several mm distance.

Endothelial Cells MAPCs express Flk1 but not CD34, PECAM, E- andP-selectin, CD36, Tie/Tek or Flt1. When MAPCs were cultured inserum-free MAPC medium with about 20 ng/mL VEGF, CD34 appeared on thecell surface and cells expressed vWF by day 14 (immunofluorescence). Inaddition, cells expressed Tie, Tek, Flk1 and Flt1, PECAM, P-selectin andE-selectin, and CD36. Results from the histochemical staining wereconfirmed by Western blot. When VEGF induced cells were cultured onmatrigel or collagen type IV, vascular tube formation was seen.

Approaches for transplantation to prevent immune rejection:

In some embodiments, it may be desired that the MAPCs (or differentiatedprogeny thereof) be treated or otherwise altered prior totransplantation/administration in order to reduce the risk ofstimulating host immunological response against the transplanted cells.Any method known in the art to reduce the risk of stimulating hostimmunological response may be employed. The following provides a fewsuch examples.

-   -   a. Universal donor cells: MAPC have cell surface profiles        consistent with evasion of immune recognition, and in their        natural state may not stimulate immune sensitization and        rejection. They may serve as natural universal donor cells even        if their progeny mature to cells which ordinarily would be        immune-recognized and rejected.

Alternatively, MAPC also can be manipulated to serve as universal donorcells. MAPCs can be modified to serve as universal donor cells byeliminating HLA-type I and HLA-type II antigens, and potentiallyintroducing the HLA-antigens from the prospective recipient to avoidthat the cells become easy targets for NK-mediated killing, or becomesusceptible to unlimited viral replication and/or malignanttransformation. Elimination of HLA-antigens can be accomplished byhomologous recombination or via introduction of point-mutations in thepromoter region or by introduction of a point mutation in the initialexon of the antigen to introduce a stop-codon, such as withchimeroplasts. Transfer of the host HLA-antigen can be achieved byretroviral, lentiviral, adeno associated virus or other viraltransduction or by transfection of the target cells with the HLA-antigencDNAs. MAPC can be used to establish and set amount or a given range orlevel of a protein in the body or blood.

-   -   b. Gene therapy: MAPCs can be extracted and isolated from the        body, grown in culture in the undifferentiated state or induced        to differentiate in culture, and genetically altered using a        variety of techniques, especially viral transduction. Uptake and        expression of genetic material is demonstrable, and expression        of foreign DNA is stable throughout development. Retroviral and        other vectors for inserting foreign DNA into stem cells are        known to those of skill in the art. (Mochizuki, H. et        al (1998) J. Virol 72(11): 8873-8883; Robbins, P. et        al (1997) J. Virol. 71(12): 9466-9474; Bierhuizen, M. et        al (1997) Blood 90(9): 3304-3315; Douglas, J. et al (1999) Hum.        Gene Ther. 10(6): 935-945; Zhang, G. et al (1996) Biochem.        Biophys. Res. Commun. 227(3): 707-711). Once transduced using a        retroviral vector, enhanced green fluorescent protein (eGFP)        expression persists in terminally differentiated muscle cells,        endothelium, and c-Kit positive cells derived from the isolated        MAPCs, demonstrating that expression of retroviral vectors        introduced into MAPC persists throughout differentiation.        Terminal differentiation was induced from cultures initiated        with 10 eGFP⁺ cells previously transduced by retroviral vector        and sorted a few weeks into the initial MAPC culture period.    -   c. Development of Hematopoietic Cells: MAPCs can be administered        (e.g., intravenously) to establish hematopoiesis (the process by        which blood cells are formed). Once the administered MAPCs have        generated blood cells and the subject has accepted the blood        type of MAPC derived blood cells, the subject may no longer        reject additional MAPCs or progeny or tissue derived therefrom.    -   d. Encapsulation: In some embodiments, the MAPCs are        encapsulated. The primary goal in encapsulation as a cell        therapy is to protect allogeneic and xenogeneic cell transplants        from destruction by the host immune response, thereby        eliminating or reducing the need for immuno-suppressive drug        therapy. Techniques for microencapsulation of cells are known to        those of skill in the art (see, for example, Chang, P., et al.,        Trends in Biotech., 17:78-83 (1999); Matthew, H. W., et al.,        ASAIO Trans, 37(3):M328-30 (1991); Yanagi, K., et al., ASAIO        Trans., 35(3):570-2 (1989); Cai Z. H., et al., Artif Organs,        12(5):388-93 (1988); Chang, T. M., Artif Organs, 16(1):71-4        (1992)). Materials for microencapsulation of cells include, for        example, polymer capsules, alginate-poly-L-lysine-alginate        microcapsules, barium poly-L-lysine alginate capsules, barium        alginate capsules, polyacrylonitrile/polyvinylchloride (PAN/PVC)        hollow fibers, and polyethersulfone (PES) hollow fibers. U.S.        Pat. No. 5,639,275, for example, describes improved devices and        methods for long-term, stable expression of a biologically        active molecule using a biocompatible capsule containing        genetically engineered cells.

Additionally, the MAPCs may be encapsulated by membranes prior toimplantation. The encapsulation provides a barrier to the host's immunesystem and inhibits graft rejection and inflammation. It is contemplatedthat any of the many methods of cell encapsulation available may beemployed. In some instances, cells are individually encapsulated. Inother instances, many cells are encapsulated within the same membrane.In embodiments in which the cells are removed following implantation,the relatively large size of a structure encapsulating many cells withina single membrane provides a convenient means for retrieval of theimplanted cells. Several methods of cell encapsulation are available tothe art, such as those described in European Patent Publication No.301,777 or U.S. Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274;5,084,350; 5,089,272; 5,578,442; 5,639,275; and 5,676,943, each of whichis incorporated herein by reference.

-   -   e. Natural Killer (NK) Cell Function: Any means which inhibits        NK cell function or depletes NK cells from a population of cells        may also be administered to prevent immune rejection, increase        engraftment and/or increase immune tolerance. Such an agent        includes an anti-NK cell antibody or irradiation or any other        material which can inhibit NK cell function or deplete NK cells        from a population.        Monitoring of Subject after Administration of MAPCs

Following transplantation, the growth and/or differentiation of theadministered MAPCs or differentiated progeny, and the therapeutic effectof the MAPCs or progeny may be monitored. Additionally, followingadministration, the immunological tolerance of the subject to the MAPCsor progeny derived therefrom may be tested by various methods known inthe art to assess the subject's immunological tolerance to MAPCs. Incases where subject tolerance of MAPCs is suboptimal (e.g., thesubject's immune system is rejecting the exogenous MAPCs), therapeuticadjunct immunosuppressive treatment, which is known in the art, of thesubject may be performed.

Genetically-Modified Stem Cells

MAPCs can be genetically altered ex vivo, eliminating one of the mostsignificant barriers for gene therapy. For example, a subject's bonemarrow aspirate is obtained, and from the aspirate MAPCs are isolated.The MAPCs are then genetically altered to express one or more desiredgene products. The MAPCs can then be screened or selected ex vivo toidentify those cells which have been successfully altered, and thesecells can be reintroduced into the subject, either locally orsystemically. Alternately, MAPCs can be genetically altered and culturedto induce differentiation to form a specific cell lineage fortransplant. In either case, the transplanted MAPCs provide astably-transfected source of cells that can express a desired geneproduct. Especially where the patient's own bone marrow aspirate is thesource of the MAPCs, this method provides an immunologically safe methodfor producing transplant cells.

Methods for Genetically Altering MAPCs

Cells isolated by the methods described herein can be geneticallymodified by introducing DNA or RNA into the cell by a variety of methodsknown to those of skill in the art. These methods are generally groupedinto four major categories: (1) viral transfer, including the use of DNAor RNA viral vectors, such as retroviruses (including lentiviruses),Simian virus 40 (SV40), adenovirus, Sindbis virus, and bovinepapillomavirus for example; (2) chemical transfer, including calciumphosphate transfection and DEAE dextran transfection methods; (3)membrane fusion transfer, using DNA-loaded membranous vesicles such asliposomes, red blood cell ghosts, and protoplasts, for example; and (4)physical transfer techniques, such as microinjection, electroporation,nucleofection or direct “naked” DNA transfer. MAPCs can be geneticallyaltered by insertion of pre-selected isolated DNA, by substitution of asegment of the cellular genome with pre-selected isolated DNA, or bydeletion of or inactivation of at least a portion of the cellular genomeof the cell. Deletion or inactivation of at least a portion of thecellular genome can be accomplished by a variety of means, including butnot limited to genetic recombination, by antisense technology (which caninclude the use of peptide nucleic acids, or PNAs), or by ribozymetechnology, for example. Insertion of one or more pre-selected DNAsequences can be accomplished by homologous recombination or by viralintegration into the host cell genome. The desired gene sequence canalso be incorporated into the cell, particularly into its nucleus, usinga plasmid expression vector and a nuclear localization sequence. Methodsfor directing polynucleotides to the nucleus have been described in theart. The genetic material can be introduced using promoters that willallow for the gene of interest to be positively or negatively inducedusing certain chemicals/drugs, to be eliminated following administrationof a given drug/chemical, or can be tagged to allow induction bychemicals (including but not limited to the tamoxifen responsive mutatedestrogen receptor) expression in specific cell compartments (includingbut not limited to the cell membrane).

Homologous recombination

Calcium phosphate transfection, which relies on precipitates of plasmidDNA/calcium ions, can be used to introduce plasmid DNA containing atarget gene or polynucleotide into isolated or cultured MAPCs. Briefly,plasmid DNA is mixed into a solution of calcium chloride, then added toa solution which has been phosphate-buffered. Once a precipitate hasformed, the solution is added directly to cultured cells. Treatment withDMSO or glycerol can be used to improve transfection efficiency, andlevels of stable transfectants can be improved usingbis-hydroxyethylamino ethanesulfonate (BES). Calcium phosphatetransfection systems are commercially available (e.g., ProFection® fromPromega Corp., Madison, Wis.).

DEAE-dextran transfection, which is also known to those of skill in theart, may be preferred over calcium phosphate transfection wheretransient transfection is desired, as it is often more efficient.

Since the MAPC are isolated cells, microinjection can be particularlyeffective for transferring genetic material into the cells. Briefly,cells are placed onto the stage of a light microscope. With the aid ofthe magnification provided by the microscope, a glass micropipette isguided into the nucleus to inject DNA or RNA. This method isadvantageous because it provides delivery of the desired geneticmaterial directly to the nucleus, avoiding both cytoplasmic andlysosomal degradation of the injected polynucleotide. This technique hasbeen used effectively to accomplish germline modification in transgenicanimals.

MAPC can also be genetically modified using electroporation. The targetDNA or RNA is added to a suspension of cultured cells. The DNA/RNA-cellsuspension is placed between two electrodes and subjected to anelectrical pulse, causing a transient permeability in the cell's outermembrane that is manifested by the appearance of pores across themembrane. The target polynucleotide enters the cell through the openpores in the membrane, and when the electric field is discontinued, thepores close in approximately one to 30 minutes.

Liposomal delivery of DNA or RNA to genetically modify the cells can beperformed using cationic liposomes, which form a stable complex with thepolynucleotide. For stabilization of the liposome complex, dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC)can be added. A recommended reagent for liposomal transfer isLipofectin® (Life Technologies, Inc.), which is commercially available.Lipofectin®, for example, is a mixture of the cationic lipidN-[1-(2,3-dioleyloyx)propyl]-N—N—N-trimethyl ammonia chloride and DOPE.Delivery of linear DNA, plasmid DNA, or RNA can be accomplished eitherin vitro or in vivo using liposomal delivery, which may be a preferredmethod due to the fact that liposomes can carry larger pieces of DNA,can generally protect the polynucleotide from degradation, and can betargeted to specific cells or tissues. A number of other deliverysystems relying on liposomal technologies are also commerciallyavailable, including Effectene™ (Qiagen), DOTAP (Roche MolecularBiochemicals), FuGene 6™ (Roche Molecular Biochemicals), andTransfectam® (Promega). Cationic lipid-mediated gene transfer efficiencycan be enhanced by incorporating purified viral or cellular envelopecomponents, such as the purified G glycoprotein of the vesicularstomatitis virus envelope (VSV-G), in the method of Abe, A., et al. (J.Virol. (1998) 72: 6159-6163).

Gene transfer techniques which have been shown effective for delivery ofDNA into primary and established mammalian cell lines usinglipopolyamine-coated DNA can be used to introduce target DNA into MAPCs.This technique is generally described by Loeffler, J. and Behr, J(Methods in Enzymology (1993) 217: 599-618).

Naked plasmid DNA can be injected directly into a tissue mass formed ofdifferentiated cells from the isolated MAPCs. This technique has beenshown to be effective in transferring plasmid DNA to skeletal muscletissue, where expression in mouse skeletal muscle has been observed formore than 19 months following a single intramuscular injection. Morerapidly dividing cells take up naked plasmid DNA more efficiently.Therefore, it is advantageous to stimulate cell division prior totreatment with plasmid DNA.

Microprojectile gene transfer can also be used to transfer genes intoMAPCs either in vitro or in vivo. The basic procedure formicroprojectile gene transfer was described by J. Wolff (GeneTherapeutics (1994) at page 195). Briefly, plasmid DNA encoding a targetgene is coated onto microbeads, usually 1-3 micron sized gold ortungsten particles. The coated particles are placed onto a carrier sheetinserted above a discharge chamber. Once discharged, the carrier sheetis accelerated toward a retaining screen. The retaining screen forms abarrier which stops further movement of the carrier sheet while allowingthe polynucleotide-coated particles to be propelled, usually by a heliumstream, toward a target surface, such as a tissue mass formed ofdifferentiated MAPCs. Microparticle injection techniques have beendescribed previously, and methods are known to those of skill in the art(see Johnston, S. A. et al (1993) Genet. Eng. (NY) 15: 225-236;Williams, R. S. et al (1991) Proc. Natl. Acad. Sci. USA 88: 2726-2730;Yang, N. S. et al (1990) Proc. Natl. Acad. Sci. USA 87: 9568-9572).

Signal peptides can be attached to plasmid DNA, as described bySebestyen et al. (Nature Biotech. (1998) 16: 80-85), to direct the DNAto the nucleus for more efficient expression.

Viral vectors are used to genetically alter MAPCs and their progeny.Viral vectors are used, as are the physical methods previouslydescribed, to deliver one or more target genes, polynucleotides,antisense molecules, or ribozyme sequences, for example, into the cells.Viral vectors and methods for using them to deliver DNA to cells arewell known to those of skill in the art. Examples of viral vectors whichcan be used to genetically alter the cells of the present inventioninclude, but are not limited to, adenoviral vectors, adeno-associatedviral vectors, retroviral vectors (including lentiviral vectors),alphaviral vectors (e.g., Sindbis vectors), and herpes virus vectors.

Retroviral vectors are effective for transducing rapidly-dividing cells,although a number of retroviral vectors have been developed toeffectively transfer DNA into non-dividing cells as well (Mochizuki, H.et al (1998) J. Virol. 72: 8873-8883). Packaging cell lines forretroviral vectors are known to those of skill in the art. Packagingcell lines provide the viral proteins needed for capsid production andvirion maturation of the viral vector. Generally, these include the gag,pol, and env retroviral genes. An appropriate packaging cell line ischosen from among the known cell lines to produce a retroviral vectorwhich is ecotropic, xenotropic, or amphotropic, providing a degree ofspecificity for retroviral vector systems.

A retroviral DNA vector is generally used with the packaging cell lineto produce the desired target sequence/vector combination within thecells. Briefly, a retroviral DNA vector is a plasmid DNA which containstwo retroviral LTRs positioned about a multicloning site and SV40promoter so that a first LTR is located 5 to the SV40 promoter, which isoperationally linked to the target gene sequence cloned into themulticloning site, followed by a 3′ second LTR. Once formed, theretroviral DNA vector can be transferred into the packaging cell lineusing calcium phosphate-mediated transfection, as previously described.Following approximately 48 hours of virus production, the viral vector,now containing the target gene sequence, is harvested.

Targeting of retroviral vectors to specific cell types was demonstratedby Martin, F., et al., (J. Virol. (1999) 73: 6923-6929), who usedsingle-chain variable fragment antibody directed against the surfaceglycoprotein high-molecular-weight melanoma-associated antigen fused tothe amphotropic murine leukemia virus envelope to target the vector todelivery the target gene to melanoma cells. Where targeted delivery isdesired, as, for example, when differentiated cells are the desiredobjects for genetic alteration, retroviral vectors fused to antibodyfragments directed to the specific markers expressed by each celllineage differentiated from the MAPCs can be used to target delivery tothose cells.

Lentiviral vectors are also used to genetically alter MAPCs. Many suchvectors have been described in the literature and are known to those ofskill in the art (Salmons, B. and Gunzburg, W. H. (1993) Hum. GeneTherapy 4: 129-141. These vectors have been effective for geneticallyaltering human hematopoietic stem cells (Sutton, R., et al (1998) J.Virol. 72: 5781-5788). Packaging cell lines have been described forlentivirus vectors (see Kafri, T., et al (1999) J. Virol. 73: 576-584;Dull, T., et al (1998) J. Virol. 72: 8463-8471).

Recombinant herpes viruses, such as herpes simplex virus type I (HSV-1)have been used successfully to target DNA delivery to cells expressingthe erythropoietin receptor (Laquerre, S., et al (1998) J. Virol. 72:9683-9697). These vectors can also be used to genetically alter theMAPCs, which the inventors have demonstrated to be stably transduced bya viral vector.

Adenoviral vectors have high transduction efficiency, can incorporateDNA inserts up to 8 Kb, and can infect both replicating anddifferentiated cells. A number of adenoviral vectors have been describedin the literature and are known to those of skill in the art (see, forexample, Davidson, B. L., et al (1993) Nature Genetics3: 219-223;Wagner, E., et al (1992) Proc. Natl. Acad. Sci. USA 89: 6099-6103).Methods for inserting target DNA into an adenovirus vector are known tothose of skill in the art of gene therapy, as are methods for usingrecombinant adenoviral vectors to introduce target DNA into specificcell types (Wold, W (1998) Humana Methods in Molecular Medicine,Blackwell Science, Ltd.). Binding affinity for certain cell types hasbeen demonstrated by modification of the viral vector fiber sequence.Adenovirus vector systems have been described which permit regulatedprotein expression in gene transfer (Molin, M., et al (1998) J. Virol.72: 8358-8361). A system has also been described for propagatingadenoviral vectors with genetically modified receptor specificities toprovide transductional targeting to specific cell types (Douglas, J., etal (1999) Nature Biotech. 17: 470-475). Recently described ovineadenovirus vectors even address the potential for interference withsuccessful gene transfer by preexisting humoral immunity (Hofmann, C.,et al (1999) J. Virol. 73: 6930-6936).

Adenovirus vectors are also available which provide targeted genetransfer and stable gene expression using molecular conjugate vectors,constructed by condensing plasmid DNA containing the target gene withpolylysine, with the polylysine linked to a replication-incompetentadenovirus. (Schwarzenberger, P., et al (1997) J. Virol. 71: 8563-8571.)

Alphavirus vectors, particularly the Sindbis virus vectors, are alsoavailable for transducing the cells of the present invention. Thesevectors are commercially available (Invitrogen, Carlsbad, Calif.) andhave been described in, for example, U.S. Pat. No. 5,843,723, as well asby Xiong, C., et al (1989) Science 243: 1188-1191; Bredenbeek, P. J., etal (1993) 6 J. Virol. 7: 6439-6446; and Frolov, I., et al (1996) Proc.Natl. Acad. Sci. USA 93: 11371-11377.

The inventors have shown that MAPC possess good transduction potentialusing the eGFP-MND lentiviral vector described by Robbins, et al. (J.Virol. (1997) 71(12): 9466-9474) and eGFP-MGF vector. Using this method,30-50% of MAPC can be transduced after a short exposure of 4.6 hours toan enhanced green fluorescent protein (eGFP) vector containingsupernatants made in PA3-17 packaging cells (an amphotropic packagingcell line derived from NIH 3T3 fibroblasts and described by Miller, A.D., and C. Buttimore (Mol. Cell. Biol. (1986) 6: 2895-2902), combinedwith protamine (8 mg/ml). Expression of eGFP persists throughout theculture of undifferentiated MAPC. In addition, transfection usinglipofectamine has been successfully used to introduce transgenes inMAPCs.

Successful transfection or transduction of target cells can bedemonstrated using genetic markers, in a technique that is known tothose of skill in the art. The green fluorescent protein of Aequoreavictoria, for example, has been shown to be an effective marker foridentifying and tracking genetically modified hematopoietic cells(Persons, D., et al (1998) Nature Medicine 4:1201-1205). Alternativeselectable markers include the β-Gal gene, the truncated nerve growthfactor receptor, drug selectable markers (including but not limited toNEO, MTX, hygromycin).

Any of these techniques can also be applied to introducing atranscriptional regulatory sequence into MAPC to activate a desiredendogenous gene. This can be done by both homologous (e.g., U.S. Pat.No. 5,641,670) or non-homologous (e.g., U.S. Pat. No. 6,602,686)recombination. These are incorporated by reference for teaching ofmethods of endogenous gene activation.

Use of Stem Cells

In the case of cardiac disease, diseases which can be treated using MAPCor progeny therefrom include, but are not limited to, myocarditis,cardiomyopathy, heart failure, damage caused by heart attacks,hypertension, atherosclerosis or heart valve dysfunction. Progeny caninclude cardiomyocytes that repopulate the injured tissue or endothelialcells that provide neo-vascularization to the tissue.

MAPC can also be administered to provide vasculature in subjectssuffering from a loss and/or function of vascularization as a result ofphysical or disease related damage. Disease states characterized by aloss of vascularization and/or function, and that benefit from methodsof the present invention include vascular conditions, such as ischemia(including ischemia-reperfusion injury), congestive heart failure,peripheral vasculature disorder, coronary vascular disease, diabeticulcers, pressure ulcers, hypertension, stroke, aneurysm, thrombosis,arrhythmia, tachycardia, or surgical or physical (e.g., wounding)trauma.

MAPCs can be used to repopulate heart muscle cells by either directinjection into the area of tissue damage or by systemic injection,allowing the cells to home to the cardiac tissues. This method can beparticularly effective if combined with angiogenesis. Both the methodsof injection and methods for promoting angiogenesis are known to thoseof skill in the art. MAPCs provide a broader differentiation range toprovide a more varied source of cells for cardiac or other tissue repairutilizing these techniques.

For the purposes described herein, either autologous or allogeneic MAPCscan be administered to a patient, either in differentiated orundifferentiated form, genetically altered or unaltered, by directinjection to a tissue site, systemically, on or around the surface of anacceptable matrix, or in combination with a pharmaceutically acceptablecarrier.

In the case of degenerative myocardial disease, MAPCs provide for bothmyocyte replacement and stimulation of angioneogenesis. Improved cardiacfunction can be indicated, for example, by increased perfusion. Thistherapy can be used as a stand-alone therapy or in conjunction withrevascularization therapies. MAPCs offer the advantage of formingvascular structures to furnish and supply blood to the emerging cardiacmuscle mass.

In one embodiment, the present invention comprises methods of increasingcardiac function in a subject by administering a suitable amount ofMAPCs to the subject, wherein MAPCs incorporate into existing cardiactissues and generate new cardiac muscle.

Cardiac diseases treatable with MAPC-based therapies include, but arenot limited to, congestive heart failure, coronary artery disease,myocardial infarction, myocardial ischemia, atherosclerosis,cardiomyopathy, cardiac arrhythmias, muscle mass abnormalities, andcongenital heart disease.

Myocardial infarction (MI) is characterized by the death of myocytes, bycoagulative necrosis, myocytolysis, contraction band necrosis, orapoptosis, resulting from a critical imbalance between the oxygen supplyand demand of the myocardium. The most common cause of MI is coronaryartery thrombosis following the rupture of atheromatous plaques inepicardial blood vessels resulting in regional myocardial ischemia. Scarformation in the infarcted region impairs cardiac function.

Cardiac injury, such as MI, promotes tissue responses that enhancemyogenesis using implanted MAPCs. Thus, administration of MAPCs can, forexample, reduce the degree of scar formation, augment ventricularfunction and compensate for weakened cardiac muscle, thereby improvingcardiac function. New muscle is thereby created within an infarctedmyocardial segment. Preferably, MAPCs can be directly infiltrated intothe zone of infarcted tissue.

MAPCs, as well as damaged cardiac tissues, secrete cytokines that havebeneficial effects, including recruitment of reparative cells (e.g.,MAPCs, hematopoietic, mesenchymal stem cells) to the damaged tissue by“homing” mechanisms and modulation of inflammatory processes. Homing ofcells that can advantageously repair myocardium, such as MAPCs, can bepreferentially induced by co-administration of cytokines. MAPCs can alsopromote angiogenesis, which further enhances tissue repair.

In the past, the prevention of progression to end stage congestive heartfailure was believed to preventable only by minimizing loss of myocytemass. Indeed, left ventricular dysfunction is the strongest predictor ofnegative outcome following MI. Current treatment focuses on limitingmyocyte death, but offers little to replace those myocytes that havealready been destroyed. Additionally, other forms of heart failureresulting from cell death/loss not associated with ischemia can not becorrected by additional perfusion, but instead require restoration offunctional muscle mass. These diseases include, but are not limited to,correction of congenital abnormalities, infective myocarditis induced byviral means (HIV, Coxsackieviruses A and B), protozoa (Chagas Disease),bacteria, and idiopathic cardiomyopathies. In addition, drugs or toxins,such as those used in chemotherapy can also induce myocyte death. Theseinclude, but are not limited to, doxirubicin, adriamycin, andcatecholamines. Bacterial toxins can also cause death of muscle tissues.Hypersensitivity myocarditis and autoimmune endocarditis is anotherdisease that can be treated advantageously using the methods of thepresent invention. SLE and Sarcoidosis may also be treated usingprogenitor cell-based therapies.

MAPC-based therapies are not limited to improvement of cardiacpathology, but can be extended to any type of degenerative musculardisorder in which the primary pathology is loss of striated muscle massand/or function. This would include, but is not limited to, musculardystrophies, trauma, and myasthenia gravis. Thus, in another embodiment,the present invention comprises methods of increasing striated muscletissue mass by contacting a suitable amount of MAPCs with existingstriated muscle tissue and generating viable striated muscle tissue.

Detection of Myocardial Engraftment

Engraftment and repopulation of striated muscle tissue by evaluatingmuscle function can be monitored using various well-known imagingtechniques such as scintigraphy, myocardial perfusion imaging, gatedcardiac blood-pool imaging, first-pass ventriculography, right-to-leftshunt detection, positron emission tomography, single photon emissioncomputed tomography, magnetic resonance imaging, harmonic phase magneticresonance imaging, echocardiography, and myocardial perfusion reserveimaging.

Cardiac scintigraphy evaluates myocardial perfusion and/or function todetect physiologic and anatomic abnormalities of the heart. There arefive major classes of cardiac scintigraphy: myocardial perfusionimaging, gated cardiac blood-pool imaging, first-pass cardiac imaging,myocardial infarction imaging, and right-to-left shunt evaluation(American College of Radiology Standard for the Performance of CardiacScintigraphy).

Myocardial perfusion imaging is primarily used to detect the presence,location, and extent of coronary artery disease by evaluating thephysiologic significance or sequelae of known or suspected coronaryartery stenosis, monitoring the effects of treatment of coronary arterydisease, including revascularization and medical therapy. Myocardialperfusion imaging is also useful for detecting acute myocardialinfarction and prognosis after infarction, for evaluating the viabilityof dysfunctional myocardium, for determining the risk of myocardialevents, and for evaluating ventricular function.

An echocardiogram is a test in which ultrasound is used to examine theheart. In addition to providing single-dimension images, known as M-modeecho that allows accurate measurement of the heart chambers, theechocardiogram also offers two-dimensional (2-D) Echo and is capable ofdisplaying a cross-sectional “slice” of the beating heart, including thechambers, valves and the major blood vessels that exit from the left andright ventricle.

Doppler is a special part of the ultrasound examination that assessesblood flow (direction and velocity). In contrast, the M-mode and 2-DEcho evaluates the size, thickness and movement of heart structures(chambers, valves, etc.). During the Doppler examination, the ultrasoundbeams will evaluate the flow of blood as it makes it way though and outof the heart. This information is presented visually on the monitor (ascolor images or grayscale tracings and also as a series of audiblesignals with a swishing or pulsating sound).

Echocardiography provides important information about, among otherstructures and functions, the size of the chambers of the heart,including the dimension or volume of the cavity and the thickness of thewalls. The appearance of the walls may also help identify certain typesof heart disease that predominantly involve the heart muscle. Pumpingfunction of the heart can also be assessed by echocardiography. One cantell if the pumping power of the heart is normal or reduced to a mild orsevere degree. This measure is known as an ejection fraction or EF. Anormal EF is around 55 to 65%. Numbers below 45% usually represent somedecrease in the pumping strength of the heart, while numbers below 30 to35% are representative of an important decrease. Echocardiography canalso identify if the heart is pumping poorly due to a condition known ascardiomyopathy, or if one or more isolated areas have depressed movementdue to prior heart attacks. Thus, echocardiography can assess thepumping ability of each chamber of the heart and also the movement ofeach visualized wall. The decreased movement, in turn, can be gradedfrom mild to severe. In extreme cases, an area affected by a heartattack may have no movement (akinesia), or may even bulge in theopposite direction (dyskinesia). The latter is seen in patients withaneurysm of the left ventricle or LV.

Echocardiography identifies the structure, thickness and movement ofeach heart valve. It can help determine if the valve is normal, scarredfrom an infection or rheumatic fever, thickened, calcified, torn, etc.It can also assess the function of prosthetic or artificial heartvalves. The additional use of Doppler helps to identify abnormal leakageacross heart valves and determine their severity. Doppler is also veryuseful in diagnosing the presence and severity of valve stenosis ornarrowing. Unlike echocardiography, Doppler follows the direction andvelocity of blood flow rather than the movement of the valve leaflets orcomponents. Thus, reversed blood direction is seen with leakages whileincreased forward velocity of flow with a characteristic pattern isnoted with valve stenosis.

Echocardiography is used to diagnose mitral valve prolapse (MVP), whileDoppler identifies whether it is associated with leakage orregurgitation of the mitral valve (MR). The presence of MR frequentlyprompts the use of antibiotics prior to any dental or non-sterilesurgical procedure. Such action helps reduce the rare complication ofvalve infection.

The volume status of blood vessels can also be monitored byechocardiography. Low blood pressure can occur in the setting of poorheart function but may also be seen when patients have a reduced volumeof circulating blood (as seen with dehydration, blood loss, use ofdiuretics or “water pill”, etc.). In many cases, the diagnosis can bemade on the basis of history, physical examination and blood tests.However, confusion may be caused when patients have a combination ofproblems. Echocardiography may help clarify the confusion. The inferiorvena cava (the major vein that returns blood from the lower half of thebody to the right atrium) is distended or increased in size in patientswith heart failure and reduced in caliber when the blood volume isreduced. Echocardiography is useful in the diagnosis of fluid in thepericardium. It also determines when the problem is severe andpotentially life threatening. Other diagnoses made by Doppler orechocardiography include congenital heart diseases, blood clots ortumors within the heart, active infection of the heart valves, abnormalelevation of pressure within the lungs, among others.

Myocardial perfusion reserve (MPR) quantifies the capacity of thecirculatory response to a maximal increase in physiological demand(Siebert, J. E., et al (2002) Proc. Intl. Soc. Mag. Reson. Med. vol.10). MPR indicates the net circulatory consequence from coronary lesionsand other vascular states, regardless of their morphological appearance,including the compensation by collateral circulation. Current perfusionacquisition methods now provide adequate temporal and spatialresolution, SNR, and first-pass contrast enhancement ratio.

MPR image calculation challenges: 1) cardiac phase shifts between stressand rest acquisitions, 2) stress-rest difference in diaphragm position,3) cardiac phase jitter that introduces variability in LV edge features.Stress-rest mismatches of myocardial anatomy and the reliability of theinput function normalization may pose the ultimate limitation of MPRimaging. Intensity correction of surface coil reception modulations isrequired to determine the input function normalization, forpost-processing automation, to improve the qualitative assessment ofcardiac perfusion images, movie loops, and time-intensity curves duringinteractive review of MPR quantitative images. Given the integrative andobjective MPR images, interactive investigation of suspicious regions inthe thresholded MPR image forms the core of perfusion examinterpretation. MPR imaging may provide quantitative, objectiveinformation to reduce variability in perfusion exam interpretation, andto document MR myocardial perfusion.

The present invention is additionally described by way of the followingillustrative, non-limiting Examples.

EXAMPLES Example 1 Isolation of Human MAPCs from Bone Marrow MononuclearCells

Bone marrow mononuclear cells were obtained from bone marrow aspiratesfrom the posterior iliac crest of >80 healthy human volunteers. Ten to100 cubic centimeters of bone marrow was obtained from each subject, asshown in Table 2, which indicates the approximate number of mononuclearcells isolated from each subject. Mononuclear cells (MNC) were obtainedfrom bone marrow by centrifugation over a Ficoll-Paque density gradient(Sigma Chemical Co, St Louis, Mo.). Bone marrow MNC were incubated withCD45 and Glycophorin A microbeads (Miltenyi Biotec, Sunnyvale, Calif.)for 15 minutes and CD45⁺/Gly-A⁺ cells removed by placing the sample infront of a SuperMACS magnet. The eluted cells are 99.5% CD45⁻/GlyA⁻.

As shown in Table 2, depletion of CD45⁺ GlyA⁺ cells resulted in recoveryof CD45⁻ GlyA⁻ cells which constituted approximately 0.05 to 0.10% ofthe total bone marrow mononuclear cells.

TABLE 2 Number of Volume mononuclear Number of Number of MAPCs of BoneBM 45⁻/GlyA⁻ (estimated by limiting Marrow (cc) cells post ficoll cellpost-MACS dilution assay, LDA) 50 100 millions 100,000 50 25  80 60,00035 25  50 14,000 10 50 100 50,000 30 10 150 75,000 30 30 100 100,000 2525  80 75,000 35 100 190 78,000 25 100 150 60,000 15 100 160 160,000 85100 317 400,000 50 100 200 150,000 70 50 160 160,000 85 50 115 150,00070 25  60 60,000 30 100 307 315,000 100 100 216 140,000 80 50 130150,000 40 100 362 190,000 60 50 190 150,000 40 100 200 185,000 100 100387 300,000 170 50 100 130,000 20 150 588 735,000 300

Cells were selected that do not express the common leukocyte antigen,CD45, or the erythroid precursor marker, glycophorin-A (GlyA).CD45⁻GlyA⁻ cells constitute 1/10³ marrow mononuclear cells. CD45⁻GlyA⁻cells were plated in wells coated with fibronectin in with 2% FCS, andEGF, PDGF-BB, dexamethasone, insulin, linoleic acid, and ascorbic acid.After 7-21 days, small clusters of adherent cells developed. Usinglimiting dilution assays, the frequency of cells giving rise to theseadherent clusters is ⅕×10³ CD45⁻GlyA⁻ cells.

When colonies appeared (about 10³ cells) cells were recovered bytrypsinization and re-plated every 3-5 days at a 1:4 dilution under thesame culture conditions. Cell densities were maintained between about2-8×10³ cells/cm². Cell doubling time was 48-60 h. Immunophenotypicanalysis by FACS of cells obtained after 10-12 cell doubling showed thatcells did not express CD31, CD34, CD36, CD38, CD45, CD50, CD62E andCD62-P, Muc18, cKit, Tie/Tek, and CD44. Cells expressed no HLA-DR orHLA-class-I and expressed low levels of β2-microglobulin. Cells stainedhighly positive with antibodies against CD10, CD13, CD49b, CD49e, CDw90,Flk1. The MAPC phenotype remained unchanged for >30 cell doublings(n=15). MAPC cultures with cells capable of proliferating beyond 30 celldoublings and differentiating to all mesodermal cell-types have beenestablished from >85% of donors, age 2-50 years. In 10 donors, MAPC wereexpanded for >50 cell doublings. When cells were cultured in serum-freemedium, also supplemented with 10 ng/mL IGF, cell doubling was slower(>60 h), but >40 cell doublings could be obtained. As was seen for cellscultured with 2% FCS without IGF, cells cultured in serum-free mediumwere HLA-class-I and CD44 negative, and could differentiate into allmesodermal phenotypes.

Telomere length of MAPC from 5 donors (age 2-50 years) cultured for 15cell doublings was between about 11-16 kB. In 3 donors, this was about 3kB longer than telomere length of blood lymphocytes obtained from thesame donors. Telomere length of cells from 1 donor evaluated after 15cell doublings, 30 cells doublings and 45 cell doublings remainedunchanged. Cytogenetic analysis of MAPC recovered after 30 celldoublings showed a normal karyotype.

Example 2 Treatment of Acute MI in Rat LAD Model

Cell Isolation and Expansion of Rat MAPC

BM and MNC from Sprague Dawley or Wistar rats were obtained and platedunder conditions similar for mMAPC. After 21-28 days, cells weredepleted of CD45⁺ cells, and the resulting CD45⁻ cells were subculturedat 10 cells/well.

Similar to mMAPC, rMAPC have been culture expanded for >100 PDs.Expansion conditions of rat MAPC culture required the addition of EGF,PDGF-BB and LIF and culture on FN, but not collagen type I, laminin orMatrigel™

Rat MAPC that had undergone 42 PDs, 72 PDs, 80 PDs, and 100 PDs, wereharvested and telomere lengths evaluated. Telomeres did not shorten inculture, as was determined by Southern blot analysis after 42 PDs, 72PDs, 80 PDs, and 100 PDs. Monthly cytogenetic analysis of rat MAPCrevealed normal karyotype.

Transduction

To demonstrate that differentiated cells were single cell-derived andMAPC are indeed “clonal” multipotent cells, cultures were made in whichMAPC had been transduced with a retroviral vector and undifferentiatedcells and their progeny were found to have the retrovirus inserted inthe same site in the genome.

Studies were done using two independently derived ROSA26 MAPC, twoC57BL/6 MAPC and one rMAPC population expanded for 40 to >90PDs, as wellas with the eGFP transduced “clonal” mouse and “clonal” rMAPC. Nodifferences were seen between eGFP transduced and untransduced cells. Ofnote, eGFP expression persisted in differentiated MAPC.

Specifically, murine and rat BMMNC cultured on FN with EGF, PDGF-BB andLIF for three weeks were transduced on two sequential days with an eGFPoncoretroviral vector. Afterwards, CD45⁺ and GlyA⁺ cells were depletedand cells sub-cultured at 10 cells/well. eGFP-transduced rat BMMNC wereexpanded for 85 PDs. Alternatively, mouse MAPC expanded for 80 PDS wereused. Subcultures of undifferentiated MAPC were generated by plating 100MAPC from cultures maintained for 75 PDs and re-expanding them to >5×10⁶cells. Expanded MAPC were induced to differentiate in vitro toendothelium, neuroectoderm and endoderm. Lineage differentiation wasshown by staining with antibodies specific for these cell types.

Use in Animal MI Model

Rat MAPC cultures used in the studies were transduced by means ofretroviral transduction with a green fluorescent protein marker. Afterthe cells were transduced they were subcloned at 10 cells/well asdescribed in PCT/US02/04652 (WO 02/064748) or PCT/US01/21387. TheGFP-marked rat cultures that were used in these studies areclonally-derived as evidenced by identification of a single retroviralintegration event in the expanded population. Moreover, as described inthe PCT application, differentiation into the various cell types wasperformed on clonally-derived MAPC.

MAPCs have previously been shown to differentiate intocardiomyocyte-like cells and induce engraftment in cardiac tissue inanimal studies (Jiang, Y. et al. (2002) Nature 418(6893): p. 41-9).Consequently, they likely hold potential to repair damage in the heartcaused by myocardial infarction, congestive heart failure and vasculardisorders.

Therapeutic benefit to repair damage in the heart caused by myocardialinfarction, congestive heart failure and vascular disorders wasevaluated in a Lewis rat model for myocardial infarction. Permanentischemia is induced by direct surgical LAD ligation. The stem cells usedin these experiments were isolated from the bone marrow ofSprague-Dawley (SD) rats and stably labeled by using a lentiviralconstruct encoding green fluorescent protein (GFP). In-vitrodifferentiation of the GFP-labeled MAPC into the endothelial(mesodermal), hepatic (endodermal), and neuronal (ectodermal) lineagesand subsequent analysis of lineage specific marker expression byquantitative PCR (qPCR) confirmed that the GFP-labeled MAPCs hadretained tri-lineage differentiation potential (FIG. 2). These stemcells were at population doubling numbers greater than 200 and displayednormal karyotypes and telomere lengths comparable to early bone marrowcultures.

To test the hypothesis that delivery of MAPCs to the myocardium in theperi-infarct region can help to improve cardiac function, Lewis ratsreceived the MAPCs immediately after LAD ligation by direct injectioninto the infarct border zone (5 injections of 400,000 cells perinjection). The study schema is shown in FIG. 3. This experiment wasdesigned to show allogeneic cell use with secondary endpoints ofbenefit.

When echocardiography was performed at 2 weeks post-MI, a significantincrease in shortening fraction was observed in those animals thatreceived direct injection of MAPCs compared to the PBS vehicle controlgroup (13.9±2.2% (n=4) vs. 24.0±6.6% (n=7), p<0.5, see FIG. 4).Shortening fraction is calculated as a percentage of end diastolic minusend systolic 2-D dimension, divided by end diastolic length.

Histological analysis of the heart tissue confirmed that all of theanimals that displayed increased shortening fractions after stem cellinjection had indeed undergone successful LAD ligation, as evidenced bythe presence of extended zones of infarcted tissue in the leftventricular wall areas of the heart (FIG. 5). In addition, for allanimals that received stem cells by direct myocardial injection, GFPpositive MAPCs were identified in infarcted heart tissue and in theinfarct border zones after immuno-fluorescence analysis (FIG. 5).

An absence of inflammatory lymphocytes in H & E sections of injectedhearts that were positive for donor cells, either in the fibroticischemia zone or in healthy myocardium was observed (FIG. 5, B-D). Inaddition, no allogeneic Ab response could be detected using an serialdilutions of plasma against either donor stem cells or Sprague-Dawleysplenocytes. Detection was performed using anti-rat kappa Ab, andcontrols against peripheral blood and isotype Ab showed the ability todetect all classes of rat immunoglobulin (data not shown). Mixedlymphocyte reactions (MLR) were performed, using collected blood cellsat time of sacrifice against irradiated stem cells or Sprague Dawleysplenocytes. No shift in peak MLR kinetics was seen in treated versuscontrol animals, consistent with a lack of immune sensitization (datanot shown). Phase II experiments include the use of rat aorticendothelial cells as a positive immune sensitizing population, andpre-immunization of recipient animals with Sprague-Dawley splenocytes asa positive control likely to show immune rejection.

These first observations are in support of the hypothesis thatadministering MAPCs to the injured heart can aid in improvement of heartfunction. This first experiment used a two-week endpoint, which is shortin terms of assessing therapeutic benefit. Subsequent experiments are inprogress with aims to evaluate dose-response characteristics andfunctional benefit assessment at a longer timescale, using a 6-weekendpoint (FIG. 6). These second phase experiments all include testingthe benefit of myocardial injection of MAPCs in acute MI models.

Immunogenicity of Human MAPC

Mesenchymal stem cells have demonstrated low in vitro immunogenicity,and the ability to engraft across in allogeneic recipients (Di Nicola, Met al (2002) Blood 99:3838-3843, Jorgensen, C. et al (2002) Gene Therapy10:928-931, Le Blanc, K. et al (2003) Scandinavian Journal of Immunology57:11-20, McIntosh, K. et al (2000) Graft 6:324-328, Tse, W. et al(2003) Transplantation 75:389-397).

FIG. 7 shows that human MAPCs exhibit low in vitro immunogenicity, and,analogous to mesenchymal stem cells, are immunosuppressive when added tootherwise potent T cell MLR (Tse, W., et al (2003) Transplantation75:389-397). Responder and stimulator cells were prepared, and MLRsperformed, according to procedures described by Tse, W. et al(Transplantation 75:389-397)). Results are consistent across all donorand responder pairs tested. Accordingly, MAPCs are useful in bothsyngeneic and allogeneic applications for the purposes described above,including providing cardiomyocytes, providing myocardium, providingcardiac muscle, providing systolic and/or diastolic function, treatingMI, treating CHF, and the like, either directly by in situdifferentiation or indirectly, by secreting factors that act on othercells to provide these results.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

What is claimed is:
 1. A method of providing cardiac muscle to a subject in need thereof, said method comprising administering to the subject, in an amount effective to provide new cardiac muscle, isolated expanded human multipotent non-ES, non-germ cells that express telomerase, have the capacity to undergo 40 cell doublings in culture and have a normal karyotype, wherein the existing cardiac muscle is damaged by disease or injury.
 2. The method of claim 1 wherein the non-embryonic, non-germ cells further express one or more of oct4, rex-1, rox-1, or sox-2.
 3. The method of either of claims 1 or 2, wherein the disease is selected from the group consisting of congestive heart failure, coronary artery disease, myocardial infarction, myocardial ischemia, atherosclerosis, cardiomyopathy, idiopathic cardiomyopathy, cardiac arrhythmias, muscular dystrophy, muscle mass abnormality, muscle degeneration, infective myocarditis, drug- or toxin-induced muscle abnormalities, hypersensitivity myocarditis, autoimmune endocarditis, and congenital heart disease.
 4. The method of claim 1 wherein the cells are derived from bone marrow.
 5. The method of claim 1 wherein the cells have undergone 10-40 cell doublings in culture.
 6. The method of claim 1 wherein the cells can differentiate into at least one cell type of at least two of the endodermal, ectodermal and mesodermal embryonic lineages.
 7. The method of claim 6 wherein the cells can differentiate into at least one cell type of each of the endodermal, ectodermal, and mesodermal embryonic lineages. 